the role of the acrb and cred genes in carbon catabolite … · 2015. 4. 22. · agda, an...

IYOF

The role of the acrB and ueD genes

in carbon catabolite repression inAs per g i I I u s n i d u I ar?s.

by

Natasha Anne Boase, B.Sc. (Hons).

A thesis submitted in total fulfilment of the requirements

for the degree of Doctor of Philosophy.

School of Molecular and Biomedical Science

Discipline of Genetics

The University of Adelaide(Faculty of Sciences)

May 2004

Table of Contents

AbstractDeclarationAcknowledgementsList of FiguresList of TablesList of Abbreviations

V

viiviiiixX

xi

1CHAPTER 1: INTR ODUCTION

1,1, A, N,DUIÁ,VS AS A MODEL ORGANISM

1.2 Cnneor,r cATABoLtTE REpRESStoN lru S. ceRrvlsl¡e1,2.1 Miglp1 .2.2 Ssn6p-Tup1 p corepressor complex

1.2.3 Snflp and GlcTp-Reg1p

1.2.4 Mechanism of carbon catabolite repression

1.3 CmsoÌ,r cATABoLtTE REPRESSIoN lru A. rulour¡rus

1.3.1 creA, phenotypic and molecular analyses

1.3.2 cre9and creC1.3.3 Signalling in carbon catabolite repression

1.3.4 creD and acrB1.3.5 Model of carbon catabolite repression

1.4 AruYnses

1.4.1 Acid-stable oc-amylases

1.4.2 Acid-unstable cx,-amylases

1.4.3 Regulation of a-amylases

1.5 AmS AND OBJECTIVES

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11

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12

CHAPTER 2: MAT RIALS AND METHODS

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2.1 Mnr¡nrnls2.1.1 Enzymes

2.1.2Radioactive isotopes and nucleic acids

2.1.3 Molecular weight markers

2.1 .4 Baclerial strains

2.1.5 A. nidulans strains

2. 1 .6 0ligonucleotides2.1.7 Vectors

2.1.8 A. nidulans DNA libraries

2.1.9 Kits and miscellaneous materials

2.2 SolunoNs, BUFFERS AND MEDTA

2.2.1 Solutions and buffers

2.2.2\'iredia

2.3 Mernoos2.3.1 Manipulation ol A. nidulans

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21

21

21

22

22

23

25

25

25

26

26

27

27

27

2.3,2 Nucleic acid isolation2,3.3 Polymerase Chain Reaction (PCR)

2,3.4 DNA sequencing2.3.5 Rapid amplification of 5' cDNA ends (5' RACE)2.3.6 DNA gel electrophoresis and Southern transfer2.3.7 RNA gel electrophoresis and Northern transfer2.3,8 Ribonuclease Protection Assays2,3.9 Radioactive labelling of probes

2.3.1 0 Bacterial-2-hybrid system2.3.11 Amylase secretion plate tests2.3.12 Sequence analyses2,3.1 3 Phylogenetic analyses

2,4 NucuolDE sEeuENcE AccESStoN NUMBERS

CHAPTER 3: CLONING AND MOLECULAR ANALYSIS OF CRED 33

27

28

28

28

28

29

29

29

30

30

31

31

32

3.1 lrurnooucloru

3.2 Clo¡ll¡lc cntDvttcoMpLEMENTATroN oF ne cntDS4pHENorypE oN AcRIFLAVINE

USING COSMIDS AND COSMID DERIVATIVES

3.2.1 The creD?4 allele is recessive to the wild-type allele3.2.2 Testing cosmids 121808, P24E11, and relevant derivativesfor complementation ol lhe creD34 mutant phenotype

3,3.3 Sequence analysis of pB3

3.3 ConrrplerE coMpLEMENTATIoN oF THE cneDS4pHENorypE

3.4 foemncATtoN oF cRrDwrnlru BAC22E203.4.1 Complementation analysis of five regions within 22Notrel1

3.5 MOLEcULAR ANALYSIS or cneD3.5.1 Sequence analysis of creD and identification of lhe creD34 mutation3.5.2 Bioinformatic analysis of creD and identificalion ol apyA3.5.3 Multiple copies ol apyA do not compensate for the absence ol creD

3.6 Pnore¡rus rHAT INTERAcT wtrn CneD n¡¡o ApvA3.6.1 Testing protein-protein interactions between CreD and CreA, ApyAand CreA

3,6.2 ldentification of hulAandtesting HulA for interactions with CreD and ApyA

3.7 Dscussrol,¡

CHAPTER 4: ECULAR CHARACTERIZA

41

43

33

3333

34

38

40

45

454749

49

49

53

55

TION AND ANALYSISOF ACRB 61

4.1 lrurnooucno¡l 61

4.2 Pneruoryptc ANALysts or acnB2, acnBl4 ¡no ncnBli 61

4.3 Tne ncn92nu¡tloN suppRESsES AspEcrs oF THE IREA, cREBnruo cnE0n,turnrutPHENOTYPES 65

4.4 Mol¡cuLAB ANALYSIS or ¡cnB 68

4.4.1 Sequence analysis of acrB

4.4.2 Molecular analysis ollhe acrB mutant alleles

4.5 PnoreIru INTERACTIONS INVOLVING ACRB

4.5.1 Screening for proteins that interact with AcrB

4.5.2 Testing AcrB for interactions with other known proteins

4.6 Drscussro¡r

CHAPTER 5: IDENTIFICATION OF ANAMYLASE CLUSTER 75

68

72

72

72

73

73

5.1 lrurnooucroru

5.2 SeOueruCE ANALYSIS OF THE AMLYASE CLUSTER

5.3 ¡øyB IS NOT PART OF THE AMYLASE CLUSTER

5.4 RecuuTION OF THE AMYLASES

5.4.1 Alpha-amylase secretion plate tests

5.5 DrscussroN

CHAPTER 6: CONCLUSIONS

75

76

82

82

83

83

89

APPENDIX A 95

REFERENCES 99

ilr

Abstract

This work describes the cloning and analysis of creD, and the characterization of the

acr9gene, two components of a regulatory network controlling carbon source utilization in the

filamentous fungus Aspergillus nidulans lhal involves ubiquitination and deubiquitination.

Preliminary characterization of a novel amylase cluster in A. nidulans was also carried out as pafi

of this project.

Carbon catabolite repression is a regulatory system which allows the utilisation of the

most easily metabolised carbon source present (usually glucose), and results in repression of the

expression of a large number of genes involved in the synthesis of various enzymes required for

the utilisation of less favoured carbon sources. ln A. nidulans three components of this regulatory

network have previously been characterizedi creA, which encodes a transcriptional repressor

protein, creB which encodes a deubiquitinating enzyme, and creO which encodes a protein that

contains five WD40-repeat motifs and a proline-rich region. CreB and CreC form a complex tn

vivo and it has been proposed that this complex acts to stabilize or modify the CreA repressor

protein by removing ubiquitin moieties that either target CreA for destruction via the 265

proteasome or alter the function.

fhe creD34 mutation had been identified as a suppressor of the effects ol lhe creC27

mutation, and it was also shown to suppress the effects of the creBlS mutation, suggesting a role

lor creD in this regulatory network.The creD gene was cloned by complementation and physically

analysed, and it encodes a protein that contains an arrestin_N and anestin-C domain, and a

PP)ff motif and two P)ff motifs known to be involved in protein-protein interactions. CreD is

similar to the Rodlp and Roglp proteins from Saccharomyces cerevisiae, and a search of the A,

nidulans genome led to the identification of apyA in A. nidulans, another arrestin-N and

arrestin_C domain containing protein with a single PP)ff motif. Rodlp and Rog3p interact with

the HECT ubiquitin ligase Rsp5p in S. cerevisiae, and so the homologue of Rsp5p was identified

in A. nidulans and designaled hulA. Both CreD and ApyA interacted with HulA, as determined by

the bacterial-2-hybrid system, with ApyA and HulA interacting more strongly than CreD and HulA.

Like creD34, lhe acrB2 mutation results in an acriflavine-resistant phenotype, and so

acrB was tested for suppression of the phenotypes due lo creA, creB and creC mutations.

Mutations in acrB result in altered utilization of various carbon sources in A. nidulans, and are

heterogeneous in nature. The effects ollhe acrB2 mutation are epistatic to those due to the creB

and creC mutations, indicating a role for AcrB in this regulatory network, The acrB gene had been

cloned via complementation and the physical analysis is repofied here, with acrB encoding a

V

protein that contains three transmembrane domains and a coiled-coil region. The coiled-coil

region of AcrB was subcloned into the bacterial-2-hybrid system for testing protein-protein

interactions, but none were identified.

An amylase cluster was identified and preliminary characterization begun by R. Murphy.

Here the sequence analysis of the cluster which contains three genes, amyA, an a-amylase,

agdA, an cx-glucosidase, and amy4, the transcriptional activator of amylolytic genes is presented.

It was shown that a previously identified amylase in A. nidulans, amyB, did not form part of this

amylase cluster.

V

Acknowledgements

Firstly, I would like to acknowledge my superuisor Dr. Joan Kelly, whose guidance and

support is greatly appreciated. Joan, you are an inspiration both in and out of the lab, and I thankyou for your candour and professionalism throughout my years here, You have set a great

example of how to deal with people, and to do what needs to be done! Thankyou also for theprompt response when reviewing this thesis. I fear I have been spoilt under your supervision!

Thankyou to Robin, whose enthusiasm and amazing knowledge in the lab is

ineplaceable, You seem to always have the answers to my questions, and provide a differentperspective and renewed hope. Another thankyou for reading parts of this thesis,

Thanks to my fellow PhD student Jai. lt has been great having you in the lab - and not inthe least for the food (!) provided and late night chats. And I love the book!! Thanks to Damien F.,

good luck with your honours year and the many adventures I am sure lie ahead,I'd also like to thank people past, who have made the yea(s) pass quicker in the lab,

including Julian, Damien 4., Louise, Lidia and Jairus. And another thankyou to Rachael, whotaught me so much in honours, I hope it has stood me in good stead,..

A blanket thankyou to the genetics discipline/department. I have enjoyed my time here,

and will miss many of you. Thanks to Michelle for the tutoring work, and for being so sociable.

Thanks to current and past (Timmis lab) members; John and Damien for making prac

demonstrating more interesting with a few footy discussions, and for the Friday night beers. Sas,

for always being there for a chaVbeer, and Coralie, for the accommodations in Lorne/Melbourneand the fun that seems to follow!

Wow, more people to thank. My fellow honours students, Janet and Mel, Marie, Jane and

Bec. l'm glad we have all stayed in touch! Marie for the monthly schnitty at the Cath, Janet for just

being you ! and Melfor keeping me sane and being such a good friend.

Thanks to the past and present members of the Alladins/Genies. A couple ofpremierships, and maybe even a wooden spoon this season @, but it has kept Tuesday nights

interesting for the last few years,... And it seems our run of injuries may f inally be over!

To my great mates Lyndall and Nat, thanks for your friendship over the last couple ofdecadesl Celebratory drinks coming up!

Thankyou to Matt and Rach, and Les and Kay, for providing such great company each

week as we watch our beloved footy team. Go the crows!!

The family unit must rate a mention. Mum and Dad, you have been so supportive for the

last 30 (!) years, I love you both and am thankful for allthat you have done. Nana and Papa, foralways encouraging me to learn and spoiling me since I was born, Nanna and Aunty Rob for all

the prayers and good wishes. Thanks to my big brother Andrew, and also to Amy and KB, twogreat chicks that I couldn't live withoutl

Thankyou to all of the Thewlis Family, who have helped me out in so many different waysfor so many years now. A big thankyou to Carmel, whose courage is an inspiration! A specialprayer for Paul and James, who we miss so very much. Thanks especially to James for makingme feel like it was coolto be a scientist.

And lastly, Simon. Hmmm, so many things, so little room. 0f course than$ou for putting

up with me being a student so long, and just for putting up with me in general O. You are my best

friend, and my biggest support. I am most looking fonruard to us doing all of those things that are

on the'\,vhen you finish uni" list... or did you think it would never happen? Thankyou for being

such a great pafiner.

vilt

List of Fiqures

Chapter 1

Figure 1.1 Lifecycle ol A. nidulans

Figure 1.2 Model of carbon catabolite repression in S. cerevisiae

Figure 1.3 Model of carbon catabolite repression in A. nidulans

Figure 1.4 Enzymatic degradation of starch

Chapter 3

Figure 3.1 The creD14 allele is recessive to the wild-type allele

Figure 3,2 Parlial map of cosmids 121808, P24811 and derivatives tested for

complementation of lhe creD74 phenotype on acriflavine

Figure 3.3 Partialcomplementation of lhe creD?4 mutant phenotype

Figure 3,4 Map of the hypothetical genes contained within pB3, and ORF of the

intergenic region pBd

Figure 3.5 Complementation ollhe creD34 mutant phenotype

Figure 3.6 A schematic representation of the hypothetical genes within plasmid 22Notrel1

Figure 3.7 Location of lhe creD74 mutation within the creD gene

Figure 3.8 Schematic representation of CreD and ApyA

Figure 3.9 Evolutionary relationships between CreD and other fungal arrestin domain

containing proteins

Figure 3.10 Determining apyA copy number in tapyA transformant strains via

Southern analysis

Figure 3,11 Multiple copies of apyAdoes not compensate for lack of creD

Figure 3.12 Evolutionary relationships between the HECT ubiquitin ligases in

S. cerevisiae and A. nidulans

Figure 3,13 lnvestigation of protein-protein interactions between CreD and HulA, and

ApyA and HulA as determined by the BacterioMatch ll bacterial-2-hybrid

system

Chapter 4

Figure 4.1 Phenotypic heterogeneity among acrB mutant alleles

Figure 4.2 Epistatic effects of lhe acrB2 mutation on creA, creB and creC mutant

phenotypes

Figure 4.3 Location of the acrB mutations within the acrB gene

Figure 4.4 Schematic of AcrB

Chapter 5

Figure 5.1 Schematic of the amylase cluster

Figure 5.2 Nucleotide sequence of the amylase cluster

Figure 5.3 Amylase activity secretion plate tests

Appendix AFigure 4.1 Nucleotide sequence of the pB3 plasmid

2

5

13

15

35

36

37

39

42

4446

48

50

54

56

62

51

52

66-6769

71

77

78-81

84

96-97

IX

List of Tables

Chapter 2

Table 2.1 Strains of used E. coliin this studyTable 2.2 Strains ol A. nidulans used in this studyTable 2.3 0ligonucleotide primers used in this studyTable2.4 Plasmids used in this study

Chapter 4

Table 4.1 Phenotypic analysis ol acrB mutant strains

Appendix ATable 4.1 Custom oligonucleotides designed for the p83 plasmid

22

22

23-2425

63

95

X

aa

A

ANGIS

[6r-szP]dATPB2H

BLAST

bp, kb

BSA

coc

CDNA

ccRC-terminalDa, kDA

dATP

dCTP

dGTP

dH20

DNA

dNTPsDTT

dTTP

EDTA

FSP

G

9' ffi9' F9' n9

IPTG

î.L, ml, plM, mM

MA

MEGA

min, hrmRNAMW

NCBI

NMR

No.

N-terminalnt

ORF

0rthologousParalogousPCR

PEG

List of Abbreviations

amino acid(s)

adenine

Australian National Genomic lnformation Service

alpha-labelled deoxyadenosine triphosphate

bacterial - 2 - hybrid system

basic local alignment search tool

base pairs, kilobase pairs

bovine serum albumin

cytosine

degrees Celsius

deoxyribonucleic acid complementary to ribonucleic acid

carbon catabolite repression

carboxyl{erminalDalton(s), kiloDaltons

2'-deoxyadenosine-5'-triphosphate

2'-deoxycytosine-5'triphosphate2'-deoxyguanosine-5 -triphosphate

distilled water

deoxyribonucleic acid

2'-deoxyn ucleotide-5'-triphosphates

dithiothreitol

2'-deoxythym id ine-5'-triphosphate

ethylenediam inetetraacetic acid

fonruard sequencing primer

guanine

gram(s), milligram(s), microgram(s), nanogram(s)

isopropyl-B-D{h iogalactoside

bacteriophage lamda

litre(s), millilitre(s), microlitre(s)

moles per litre, millimoles per litre

milliAmperes

Molecular Evolutionary Genetics Analysis

minute(s), hou(s)messenger ribonucleic acid

molecular weight

National Center of Biotechnology lnformation

nitrogen metabolite repression

number

amino{erminalnucleotide(s)

open reading frame

Two genes are orthologous if they diverged after a speciation event

Two genes are paralogous if they diverged after a duplication event

polymerase chain reactionpolyethylene glycol

X

5'.RACE@

RNArRNA

RSP

SDS

sscTM

T

TAE

Taq

TE

TrisU

Ub

UTR

UV

X-gal

%(vlvl% (wlvl

rapid amplification of 5' cDNA endsregistered

ribonucleic acid

ribosomal RNA

reverse sequencing primer

sodium dodecyl sulphate

saline sodium citrate

Trademark

thymidine

Tris-acetate EDTA

Thermus aquaticus

Tris-EDTA

Tris[hydroxymethyl] amino methane

Unit(s) of enzyme

ubiquitin

untranscribed region

ultraviolet

5'-bromo-4-ch loro-3-indoyl-B-D-galactopyranosidepercent volume per volumepercent weight per volume

xil

Chapter 1 : lntroduction

CHAPTER 1: INTRO DU CTION

Many microorganisms are able to adapt to utilise a number of nutritional substrates to

obtain carbon and nitrogen, and have developed mechanisms to regulate gene expression in

response to their environment. ln the filamentous fungus Aspergillus nidulans the preferred

source of energy is the oxidation of glucose, and in the presence of a variety of potential carbon

sources glucose is used preferentially to less readily metabolised carbon sources. Carbon

metabolism is adjusted according to the immediate availability of glucose by the general process

of carbon catabolite repression, and also via induction by pathway specific regulatory genes.

Carbon catabolite repression is a wide domain regulatory system that responds to the

local nutritional environment to regulate carbon metabolism. ln the presence of the preferred

carbon source glucose, the structural genes that encode enzymes and permeases for utilising

alternative sources of carbon such as ethanol, proline, acetamide and starch are repressed, Upon

depletion of glucose these pathway specific enzymes are expressed to utilize the specific

alternative carbon source present in the environment. Hence the organism conserves energy by

only producing proteins as they are required. Carbon catabolite repression has been identified in

bacteria and fungi, and has been most extensively studied in the yeast Saccharomyces

cerevisiae.

1.1 A. nidulans as a model organism

A. nidulans is an ascomycete fungus that has proven to be a very successful

experimental organism in which to study gene regulation and, in particular, carbon catabolite

repression. A. nidulans has the ability to grow on simple defined solid media as compact colonies,

with most strains producing mature colonies within 48 hours on defined medium, as well as the

ability to grow in submerged culture. A. nidulans has both mitotic asexual and meiotic sexual life

cycles, plus a parasexual cycle that can be manipulated under laboratory conditions to induce

strains to cross (Figure 1.1), A. nidulans has a haploid genome under normal circumstances,

allowing for the direct observation of recessive phenotypes. Diploids and heterokaryons can be

forced from complementing auxotrophs (strains with different nutritional requirements from each

other), and there is heterokaryon compatibility between all laboratory strains as they derived from

a single source. Maintenance ol A. nidulans can be by asexual reproduction via mycelial

propagation, or through the germination of conidia. Large numbers of A. nidulans colonies can be

screened easily in both a practical and economic sense, with strong selective techniques, The

ease of genetic manipulation, the ability to perform mutagenesis, recombination,

1


.æ# n[lr fo*ÀÊ1ori

4sú{)5P0Éßnuclçtr rxefr¿11$e

hyÊhål Èn|¡loroliå

ÐrÉ Lðlo

t\

ft$fllll{r. rtfirt

MAìÐ4ê CÐI.ÔNY

ctEls foTlJsctu$

ffi @rlilf tÀ¡. çÐNIÐIIJ[J ßçHlùt0pl¡0nÈ

Figure 1.1 Lifecycle of A. nidulans.

Figure taken from Figure 4.1 , Martinelli (1994)

2


complementation and dominance tests, and the efficiency of transformation (homologous and

heterologous) make A. nidulans an ideal model system for the study of gene regulation. The

sequence of the 30.1 Mb A, nidulans genome has recently been publicly released,

complementing genetic mapping (Clutterbuck, 1997) and allowing bioinformatic analyses. A.

nidulans has well documented biochemical pathways, under the regulation of pathway specific

and global regulatory controls, permitting the investigation of regulatory networks.

1.2 Carbon catabolite repression in S. cerevisiae

Carbon catabolite repression has been extensively studied in the yeast S. cerevisiae, and

the mechanisms involved have been well elucidated. The central components of carbon

catabolite repression can be classified into positive regulators (i.e. the Snflp protein kinase

complex), and negative regulators of carbon source utilization (i.e. the Miglp transcriptional

repressor, the Ssn6p-Tup1p corepressor complex and the Reglp-Glc7p protein phosphatase 1),

as detailed below.

1.2.1 Miglp

MlGl encodes a transcriptional repressor containing two DNA binding CyszHisz zinc-

fingers in the amino terminal region, a nuclear exporl signal and a nuclear localization signal

(Nehlin and Ronne, 1990). Migl p binds DNA at the consensus sequence (5'-

WWWWWNSYGGGG-3'), found in carbon catabolite repressible promoters (Nehlin and Ronne,

1990). ln the presence of high levels of glucose Miglp rapidly moves into the nucleus where it is

able to bind to the promoters of glucose-repressible genes. Miglp recruits the Ssn6p-Tup1p

corepressor complex to the promoters of many glucose-repressed genes to effect repression

(Treiteland Carlson, 1995). Upon depletion of glucose, Miglp is phosphorylated, dissociates with

the corepressor complex and is exported from the nucleus to the cytoplasm via the nuclear

exportin MsnSp (DeVit et al., 1997; DeVit and Johnston, 1998). This glucose dependant sub-

cellular localization of Migl p responds quite rapidly to the presence/absence of glucose.

1.2.2 Ssn6p-Tup1 p corepressor complex

Tuplp is a pleiotropic repressor containing six WD40 repeats (mediating protein-protein

interactions), and Ssn6p is a pleiotropic repressor containing ten tetratricopeptide repeats (TPR),

These two proteins associate in a high molecular weight corepressor complex, composed of one

Ssn6p subunit and multiple Tuplp subunits (Varanasi et al., 1996; Redd et al., 1997). This

corepressor complex utilizes different DNA binding proteins to repress transcription of genes

regulated not only by glucose (via Mig1p, see above), but also by DNA damage, oxygen and cell

3


type, Distinct combinations of TPR motifs are required for Ssn6p to interact with these specific

DNA binding proteins, and Ssn6p acts as a linker between the pathway specific DNA binding

proteins and the Tuplp transcriptional repressor (Tzamarias & Struhl, 1995). The tethering of the

corepressor complex to the promoter allows Tupl to interact with histones H3 and H4, stabilizing

adjacent nucleosomes and blocking the transcription machinery access to the promoter, thereby

effecting repression (Edmendson etal., 1996).

1.2.3 Snflp and GlcTp-Reg1p

Snflp is a serineithreonine protein kinase that is highly conserved among eukaryotes

(Celenza and Carlson, 1986, 1989). Snflp is parl of a high molecular weight complex, including

scaffolding proteins Sip1p, Sip2p and Gal83p, and complexes with Snf4p (Jiang and Carlson,

1997). ln the presence of glucose, the Snflp kinase is inactive, allowing Miglp to affect

repression. Snflp contains a regulatory domain and a catalytic domain, and in the presence of

glucose the regulatory domain of Snflp autoinhibits the catalytic domain of Snf1p. ln low or no

glucose conditions, a conformational change results in the regulatory domain of Snf 1p binding to

Snf4p directly, freeing the catalytic domain of Snflp to phosphorylate Miglp (Jiang and Carlson,

1996). Ludin ef a/. (1998) proposed that the Snflp kinase is phosphorylated to activate the

catalytic activity of the Snf 1p complex by an as yet unidentified kinase. The GlcTp-Reg1p protein

phosphatase I responds to increasing levels of glucose by interacting with the active Snf 1p

complex, inducing a conformational change in the Snf 1p complex back to the autoinhibited state,

possibly by dephosphorylating the complex (Ludin ef a/., 1998),

1.2.4 Mechanism of carbon catabolite repression

ln the presence of high glucose (the repression signal), the Miglp repressor protein is

imported into the nucleus and binds at glucose-repressible promoters to effect repression by

recruiting the Ssn6p-Tup1p corepressor complex (Figure 1.2). The Snflp kinase that acts to

phosphorylate Miglp is inactive in high glucose conditions. Upon depletion of glucose (the

derepression signal), the Snf 1p kinase becomes active, due to the Snf4p activating subunit

binding to the regulatory subunit of Snf1p, allowing the catalytic subunit of Snflp to function.

Snflp phosphorylates Mig1p, resulting in the exportof Miglp from the nucleus to the cytoplasm,

allowing expression of glucose repressible-genes.

The exact glucose (repressing) signal that affects the Snf 1p complex catalytic activity is

yet to be elucidated. Three kinases found in S. cerevisiae, hexokinases Hxk1p, Hxk2p and

glucokinase Glk1p, are responsible for the phosphorylation of hexose sugars as the first

4


snþ

repr+ssionsign*l

cytoplasm

nudeu$

Figure 1.2 Model of carbon catabolite repression in S. cerevisiae.NES Nuclear Exporl SequenceNLS Nuclear Localization SequenceURE Upstream Regulatory Element i.e.Mig1p binding site

Figure adapted from Figure 4, Schuller (2003).

5


irreversible step in glucose metabolism, These three kinases are expressed differentially (Herrero

et al., 1995) and are required for shorl{erm glucose repression, but only Hxk2p is specifically

required for long-term glucose repression (De Winde et a\.,1996). The catalytic activity of Hxk2p

appears to be independent of its function in glucose signalling (Mayordomo and Sanz,2001).

Hxk2p is found in the cytoplasm and nucleus, and this nuclear localisation is necessary for

glucose repression signalling (Herrero et al., 1998), Hxk2p was shown to form part of a DNA

binding complex at the SUC2 (glucose-repressible) promoter to establish glucose repression

(Herrero et a\.,1998), The phosphorylation status of Hxk2p also appears to be important for its

function(Randez-Gil ef a/., 1998).Sanz etal.(2000) identifiedaroleforHxk2pinregulatingthe

activity of Snf 1p via the phosphorylation of Reg1p, the regulatory subunit of protein phosphatase I

(see Section 1.2.3), although the exact mechanism remains unknown. Hxk2p appears to have a

central role in glucose sensing via the main glucose repression pathway, but the actual glucose

signal that triggers repression is yet to be determined.

1.3 Carbon catabol¡te repression in A, nidulans

S. cerevisiae is a fermentative yeast that is highly adapted for growth in sugar rich

environments, and has evolved to utilize glucose differently from most other organisms, preferring

fermentation of glucose to ethanol rather than the oxidation of glucose, even under aerobic

conditions. A. nidulans metabolizes glucose like most other eukaryotic organisms, undergoing

aerobic oxidation via the mitochondrial pathways, and as such the regulatory system of carbon

catabolite repression may be quite different to that found in S. cerevisiae.

ln order to identify regulatory proteins that affect carbon catabolite repression in A.

nidulans a genetic screen that exploits the interaction between carbon catabolite repression and

nitrogen metabolite repression was devised (Arst & Cove, 1973). Nitrogen metabolite repression

is a regulatory system whereby prefened nitrogen sources such as ammonium or glutamine are

utilised preferentially to less favoured nitrogen sources, and is mediated by the positively acting

regulatory gene areA (Arst & Cove, 1973). ln the presence of glucose, and absence of ammonia

or glutamine, the transcriptional activator protein AreA is required for the expression of genes

required for the catabolism of alternative nitrogen sources, ln the presence of glucose null alleles

of areA are unable to grow on any nitrogen source other than ammonium as these strains cannot

derepress genes that are subject to nitrogen metabolite repression. Some alternative carbon

sources, such as proline and acetamide, can also serve as alternative nitrogen sources. The

enzymes required for the metabolism of such compounds are therefore controlled by both carbon

catabolite repression and nitrogen metabolite repression, and the relief of either repression leads

6


to expression. ln the absence of glucose areA lack of function strains can grow on medium

containing acetamide or proline as the only sources of carbon and nitrogen, but not in the

presence of glucose. Arst and Cove (1973) exploited this interaction to identify mutations that

suppress the effects of loss of function are,4 alleles such that the enzymes for acetamide or

proline utilization are produced even in the presence of glucose (carbon catabolite repressing

conditions). This genetic screen was used to identify regulatory proteins affecting carbon

catabolite repression, encoded by the creA, creBand creC genes (Arst and Cove, 1973; Bailey

and Arst, 1975, Hynes and Kelly, 1977).

1.3.1 creA, phenotypic and molecular analyses

The creA mutations constitute the majority of mutants identified by the genetic screen

described above, and result in varying levels of derepression (i.e. inappropriate expression of

genes that would normally be repressed) of a wide range of genes. For example , creA3}leads to

high levels of derepression of acetamidase (AmdS) and alcohol dehydrogenase I (Adhl), but only

very low levels of derepression of enzymes for proline utilization. 0n the other hand, creA220

leads to high levels of derepression of enzymes for proline and acetamide use, but only very low

levels of derepression of alcohol dehydrogenase I (Shroff et a\.,1997), The general trend towards

derepression is obserued in all mutant creA alleles, but individual pathways subject to carbon

catabolite repression show a different spectrum of affects, -lhe creA mutant alleles exhibit

heterogeneous, pleiotropic phenotypes indicating a regulatory rolelor creA.

fhe creA gene in A. nidulans has been cloned and characterised (Dowzer and Kelly,

1989, 1991). The derived polypeptide of 416 aa contains several features characteristic of a

transcriptional repressor, such as two zinc{ingers of the CyszHisz class, an alanine rich region,

and frequently appearing (STPXX) motifs. The consensus sequence bound by the CreA zinc

finger domain is (5'-SYGGRG-3'), and the presence of an AT-rich sequence 5'to the consensus

sequence can affect the binding of some of the sequences that fit this consensus (Kulmberg et

a/., 1993; Cubero and Scazzocchio, 1994). Towards the C{erminal end of the protein a stretch of

about 40 amino acids is completely conserved between a number of filamentous fungi and is

similar to the Rgrl p repressor protein in S. cerevrsrae (Drysdale et al., 1993), However this Rgrl -

like region is not functionally conserved with the yeast protein as the replacement of the Rgrl -like

region in CreA by the corresponding Rgrl region from S. cerevisiae failed to complement (Shroff,

1 ee7).

Mutant creA alleles can be grouped into two broad classes, missense mutations in the

DNA binding region, and frameshift or nonsense mutations that result in a truncated CreA

7


peptide. A null allele was constructed for creA,and this shared a similar mutant phenotype to a

number of other identified mutations that resulted in truncated CreA protein (Shroff et a\.,1997).

The most morphologically extreme mutation identified, which led to a very compact morphology,

slow growth and a high degree of derepression, is creÁ306 that disrupts the recognition cr-helix of

the second zinc finger in the DNA binding domain. The creÁ306 allele is the only allele predicted

to produce a full-length protein where DNA binding is completely abolished, and thus the extreme

phenotype could be due to the CreA306 protein titrating other proteins that interact with CreA

(Shroff et a\.,1997).

CreA mediates carbon catabolite repression for a number of alternative carbon utilising

systems, and as such can be considered a global regulator. There appears to be a role for CreA

in both repressing (presence of glucose) and nonrepressing (absence of glucose) conditions, as

indicated by elevated levels of Adhl in creA mutants under nonrepressing (+/- induced) conditions

(Mathieu and Felenbok, 1994; Shroff et a1.,1996). Northern and mutational analyses indicate that

lhe creA gene itself is autoregulated, and it contains CreA binding sites in its own promoter

(Shroff ef a/., 1996; Strauss ef a/., 1999). ln addition to transcriptional regulation, Strauss et al.

(1999) speculated that the CreA protein could be subject to regulation via degradation or protein

inactivation under derepressing conditions as there was no efficient CreA binding to consensus

oligomers in an electrophoretic mobility shift assay under these conditions.

The CreA protein has been identified in a large number of fungi, including Aspergillus

oryzae, Aspergillus niger, Sclerotinia sclerotiorum and Trichoderma reesei(reviewed in Kelly,

2004). The zinc-finger region of CreA shows strong sequence similarity to the zinc{inger region

of the S. cerevisiae Miglp repressor (see Section 1.2.1). Crel from S. sclerotiorum is functionally

related to CreA from A. nidulans, but cannot complement MlGl and MIG2 deliciencies in S.

cerevisiae (Vautard-Mey et al., 1999), Vautard-Mey et al. (1999) raised polyclonal antibodies

against a fusion protein, GST-Cre1, and demonstrated by subcellular fractionation that Crel is

localised in the nuclei of glucose-grown hyphae, and in the cytoplasm when glucose is removed

from the culture medium, similartothe glucose-dependant localisation of Miglp in S. cerevisiae,

Mutation of a putative AMPK phosphorylation site in CreA (Ser266) abolishes the repressor

activity of the fusion GST-Cre1 protein, but its nuclear targeting was not affected (Vautard-Mey &

Fevre, 2000), Phosphorylation has also been implicated in the DNA binding of Crel from

Hypocrea jecorinalTrichoderma reesei, with the phosphorylation of Ser241 required to allow a

DNA binding conformation of Crel (Cziferszky et a1.,2002), A phosphorylation-independent DNA

binding mutation (S2414) resulted in the permanent carbon catabolite repression of

cellobiohydrolase 1 expression (Cziferszky et a1.,2002). Thus phosphorylation of Crel results in

B


the ability to effect repression via DNA binding, whereas it relieves S. cerevisiae from Migl

dependant repression by signalling export from the nucleus.

1.3.2 creB and creC

The genetic screen that identified the involvement of creAin carbon catabolite repression

also identified the creB and creC genes as suppressors of areA2lTon medium contain glucose

and acetamide. creB has previously been described as the molB mutation that confened

resistance to toxic concentrations of molybdate (Arst et a|.,1970; Arst, 1981). fhe creB and creC

mutants share an almost identical phenotype, including resistance to molybdate and sensitivity to

acrif lavine in complete media. Mutations in creB and creC result in the derepression of a range of

enzymes, such as acetamidase and alcohol dehydrogenase, that are usually subject to carbon

catabolite repression, but the pathways affected are only a subset of those systems affected by

mutations in creA (Hynes & Kelly, 1977).ln addition, cre9and cre0mutations also lead to the

failure to derepress enzymes for the metabolism of a range of carbon sources including quinate

and proline, and lead to elevated levels of enzymes such as extracellular proteases in the

presence or absence of glucose, indicating a role for CreB and CreC in both repressing and

derepressing conditions (Hynes & Kelly, 1977). The shared pleiotropic mutant phenotype ol creB

and creC strains indicated that these genes may be regulatory, and the creB creC double mutant

phenotype is not additive, indicating that these proteins may be pafi of a protein complex or act in

the same pathway (Hynes & Kelly, 1977). There is no heterogeneity between different mutant

alleles.

fhe creB gene was cloned utilising the linkage ol cre9lo acoB, with a chromosome walk

along chromosome ll leading to the identification of a complementing cosmid. The creB gene

encodes a767 aa protein that is a functionaldeubiquitinating enzyme (Lockington & Kelly, 2001).

CreB contains six deubiquitination (DUB) homolog domains, indicative of members of the

ubiquitin processing protease (ubp) family, a coiled-coil region, most likely to be involved in

substrate recognition, and four predicted PEST sequences, which are implicated as signals for

proteolysis. There are highly conserved proteins to CreB in humans (UBH1\, Arabidopsis thaliana

(UBP3), Pichia anomala (UBP1), and hypothetical proteins trom Drosophila melanogaster

(44F56066) and Caenorhabditis elegans (CAB542B6), but interestingly no similar sequence in S.

cerevisiae was identif ied,

fhe creC gene was cloned by complementation utilising its proximity to the g/nA gene

(Todd ef al., 2000). creC encodes a 630 aa protein that contains five WD40 repeats, motifs

involved in protein-protein interactions, a proline rich region, and a putative nuclear localisation

I


signal (Todd et a\.,2000). Highly conserved proteins are found in Schizosaccharomyces pombe

(Yde3), mouse (DMR-N9), humans (DMR-N9), A. thaliana (T2N18,8) and C. elegans (C0886.7).

DMR-N9 in mouse and humans is associated with the myotonic dystrophy region (Mahadevan ef

a/., 1993; Shaw ef a/., 1993). Again it is significant that there is no closely conserved CreC

sequence in S. cerevrsrae, although the WD40 region does show weak similarity with Tuplp (see

Section 1.2.2). However the RcoA protein from A. nidulans shows considerably higher similarity

to Tupl p than does CreC, and deletion ol rcoA does not affect carbon catabolite repression

(Hicks et a\.,2001). RcoA is involved in asexual development and sterigmatocystin production

and the different function of the Tupl homologue in A. nidulans compared to that in S. cerevisiae

implies that a different mechanism of carbon catabolite repression exists in A. nidulans (Hicks ef

a1.,2001), The presence of CreB and CreC homologues in mouse and humans, but not in S.

cerevisiae, supporls that the regulatory mechanism CreB and CreC are involved in is conserved

among multicellular eukaryotes but is not found in S. cerevisiae.

CreB and CreC have been shown to be present in a complex in vivo, and it has been

proposed that the CreB/CreO deubiquitination complex removes ubiquitin moieties from CreA and

other substrates, thereby modifying or stabilising these proteins (Lockington & Kelly, 2002). That

CreB and CreC proteins are present in a protein complex in vivo under both repressing and

derepressing conditions was shown by co-immunoprecipitation experiments (Lockington & Kelly,

2002). Overexpression of the CreB deubiquitination enzyme can compensate for the lack of the

CreC, but nol vice versa, indicating that CreB acts downstream of CreC (Lockington & Kelly,

2002). The role of CreC may be to stabilize or activate the CreB deubiquitination enzyme,

perhaps by CreC masking the PEST region of CreB to stabilize or alter substrate recognition. The

presence of proteins in a range of organisms strongly conserved with the CreB and CreC proteins

identified in A. nidulans implies that these proteins may be a component of a regulatory network

that is present in most eukaryotes.

Protein stability is a key regulatory mechanism in the control of metabolism, the cell

cycle, cell growth and development, and disease. Selective degradation or stabilization of

intracellular proteins by ubiquitin-dependent pathways is essential for conect regulation of many

cellular processes, Protein ubiquitination is highly conserved amongst eukaryotes and involves

the addition of the 76 amino acid peptide ubiquitin (Ub) to protein substrates. lt entails a cascade

of reactions involving three enzyme complexes: E1 (ubiquitin-activating enzymes), E2 (ubiquitin-

conjugating enzymes) and E3 (ubiquitin-protein ligases) which act sequentially. There are a

number of conjugating enzymes and protein ligases in the cell, and various combinations confer

substrate specificity on the system, The best known role of ubiquitination is the marking of

10


proteins for destruction via the 265 proteasome, but ubiquitination can also promote endocytosis,

membrane transporl and transcriptional regulation (reviewed in Muratani and Tansey, 2003), This

process can be opposed by the action of deubiquitinating enzymes that remove ubiquitin from

specific substrates, thus stabilizing them. Perhaps the best-studied example of a regulatory

deubiquitinating enzyme in a multicellular eukaryote is the D. melanogaster Fat facets

deubiquitinating enzyme, which is required for correct eye development (Wu et a1.,1999). Genetic

analysis has revealed that the critical substrate of Fat facets in the eye is Liquid facets (Cadavid

et a1.,2000), Another example of a regulatory deubiquitinating enzyme is murine DUB-1, which

regulates cellular growth pathways by controlling the level of expression or ubiquitination state of

protein regulators at the G1/S phase transition (Zhu et a/,, 1996), although the substrates still

remain unknown.

1.3.3 Signalling in carbon catabol¡te repression

Recently Flipphi et al. (2003) investigated the role of hexose phosphorylating enzymes in

the signalling of carbon catabolite repression in A. nidulans. Transcriptional repression by glucose

was fully retained in strains lacking either hexokinase (hxkA1) or glucokinase (glkA4), but the

double hexose kinase mutants (hxkAl/glkA4) showed transcriptional derepression in the

presence of glucose and a complete absence of glucose phosphorylating activity (Flipphi ef a/.,

2003). So unlike the Hxk2p hexokinase in S. cerevlslae (see Section 1.2.4), neither hexose

kinase in A. nidulans exhibits a unique regulatory function in carbon catabolite repression (Flipphi

et a1.,2003).

1.3.4 creD and acrB

The creD?4 mutation was identified as a suppressor ollhe creC2Zmutant phenotype on

glucose medium containing fluoroacetamide, and was also found to suppress lhe creBl5 mutant

phenotype on this medium (Kelly & Hynes, 1977). The creD34 mutation suppressed other

aspects of the creB and creC mutant phenotypes, such as the derepression of lhe facAand alcA

genes as analysed through the fluoroacetate and allyl alcohol sensitivity found in creBl5 and

creC27 strains (Kelly & Hynes, 1977).ln a wild-type backgroundlhe creD34 mutation is more

resistant than wild-type on glucose and fluoroacetate medium, suggesting that the creD34

mutation leads to tighter repression of enzymes subject to carbon catabolite repression. The

suppression of the creB and creC mutant phenotypes by creD74 implies lhal creD is involved in

an opposing process to the deubiquitination role of the CreB/CreC complex, such as the

ubiquitination of the same target proteins. The creD14 mutation results in resistance to the

11


presence of acriflavine in complete medium, and sensitivity to the presence of molybdate, the

reverse of the phenotype conferred by the creA, creB and creC mutations.

The acrt2 mutation was identified as a spontaneous resistant sector that allowed growth

on complete medium containing acriflavine in a genetic screen with the joint aims of

understanding acriflavine toxicity and obtaining extra tools for genetic mapping (Roper & Kafer,

1957). Not only did mutationsin acrB result in acriflavine resistance, but also greater resistance

than wild-type to the presence of malachite green and crystal violet. Arst (1981)pointed out the

conserved genetic linkage of creBto acrB, and creC to creD, and suggested the possibility that

the regions arose as a result of duplication. The molecular nature of creB and creC makes this

origin unlikely (Todd et a1.,2000; Lockington and Kelly, 2001). However, the fact lhal creD34

suppresses phenotypes due to creB and creC, andthat acrB shares some phenotypes with creD,

indicates lhal acrB and creD may encode proteins in a process opposing deubiquitination.

1.3.5 Model of carbon catabolite repression

A model for the interaction of CreA, CreB and CreC in carbon catabolite repression has

been proposed (Lockington & Kelly, 2002) (Figure 1.3). ln the presence of glucose (i.e. under

repressing conditions), it is known that the CreA protein binds the consensus sequence in the

promoters of genes that encode enzymes/permeases that allow the utilization of alternative

carbon sources to effect carbon catabolite repression, CreA is recognised as a substrate of the

CreB deubiquitinating enzyme via the coiled-coil region of CreB, and free CreB acts to remove

ubiquitin chains from CreA, thereby stabilizing the CreA protein and allowing repression to occur,

ln the absence of glucose (i.e. under derepressing condilions), free CreB does not recognise

CreA and the PEST sites of CreB are exposed, leading to the degradation of CreB via a PEST-

mediated pathway. CreA remains ubiquitinated and is either degraded via the 265 proteasome,

or is modified and no longer binds to the promoter. ln both the presence and absence of glucose,

there is some CreB complexed with CreC, which has a role in the turnover of

enzymesipermeases required for proline and quinate utilization as an example.

Carbon catabolite repression in A. nidulans involves a regulatory de/ubiquitination

network, which is not present in S. cerevisiae, but is present in higher eukaryotes, highlighting the

impofiance of using A. nidulans to further dissect this conserued regulatory ubiquitination

network.

12


CreB

Repression

Ubiquitination Deubiquitination

Derepression

Ubiquitination Deubiquitination

Ubiquitinating

complex

Ubiquitinating

complex

Targets: Targets J\- /Ub

Ub

Ub

Outcome:

Active CreA, PrnBOutcome:

No active CreA; Active PrnB

Figure 1.3 Model of carbon catabolite repression in A. nidulans.

Figure adapted from Lockington and Kelly (2002)

CreB

CreA CreA

13

Chaoter 1 : lntroduction

1.4 Amylases

Starch is a major storage polysaccharide in plants consisting of cr-1,4-glucan chains with

o-1,6 branching. Filamentous fungi produce cx-amylases, glucoamylases and cr-glucosidases

that act in synergy to hydrolyse starch to glucose. The major group of starch hydrolysing

enzymes are o-amylases (EC 3.2.1.1, 1,4-ø-glucan glucanohydrolase), which are often

secreted. oc-amylases are the first group of starch-degrading enzymes to act on starch by

catalysing the random internal endoamylolytic cleavage of o-1,4-glycosidic bonds, resulting in

the production of glucose, maltose and other branched oligosaccharides from their substrates.

Starch is subsequently degraded by glucoamylase and cx-glucosidase. Glucoamylase (EC

3.2.1.3, glucan 1,4-o-glucosidase), and o-glucosidase (EC 3,2.1.20, also known as maltase) are

both exoamylases that cleave the cr-1,4-glycosidic bonds from non-reducing ends, and some

glucoamylases are also capable of hydrolysing the branching o-1,6-glycosidic bonds when the

next bond is an o-1,4 glycosidic bond (Figure 1.4). Starch serues as an alternative carbon

source, so carbon catabolite repression would be expected to play an important role in the

breakdown of starch, and therefore in the regulation of cr-amylase activity and the other

amylolytic enzymes that work cooperatively to reduce starch to glucose so that it can be utilised

as a carbon source.

Alpha-amylases act not only on starch, but also on glycogen and related polysaccharides

and oligosaccharides, to catalyse the random cleavage of internal o-1,4-glycosidic bonds. This

results in the production of glucose, maltose, dextrins and other branched oligosaccharides from

their substrates, which can then be further reduced. There are two major types of a-amylase

activities, acid-unstable (neutral) and acid-stable o-amylases (Minoda and Yamada, 1963).

Many of the physiochemical properties of these two types are similar, but the acid-stable

a-amylase protein retains enzymatic activity after treatment with acid, and acid-unstable

amylases do not. Structurally, the separate domains of the different amylase types are the same,

but there is a rotation of the COOH-terminal domain in acid-unstable cr-amylases relative to the

acid-stable enzyme (Boel ef a/,, 1990). Acid-unstable cr-amylases have been detected and

characterised in a number ol Aspergillus species, whereas characterisation of the acid-stable

form is limited.

14


l.'()lí

tl l glucoamylaseli¡ i¡nch

Ita)

ÕfiI (j

¡l i)tÍr-,(llt ì-, (,,t t

Ìr tf (.Ì (i!:ii:a i; !

.,--t¡ i{ I.l iill cri ií (.)t Iij

i{ cit il (iii C}IInon-reducing

end

ø.-amylasecr.-glucosídase

Figure 1.4 Enzymatic degradation of starch. The position of action of the starch-

degrading enzymes o(-amylase, glucoamylase and o(-glucosidase, o(-amylase cleaves

internal cr-1,4-glycosidic bonds; o(-glucosidase cleaves external o-1,4-glycosidic bonds;

glucoamylase cleaves external cr-1,4-glycosidic bonds, and o-1,6-glycosidic bonds when

adjacent to an o-1,4-glycosidic bond.

Figure adapted from Stryer (1998)

15


1.4.1 Acid-stable a-amylases

Acid-stable cr-amylases were first detected after the purified crystalline form of o-

amylase from A. nigerwas treated with acid (pH 2.5) at 37"C for 30 minutes and retained some

amylolytic activity (Minoda and Yamada, 1963). The catalytic action of acid-stable and acid-

unstable cr-amylases is quite similar, but the dextrinizing unit per mg enzyme protein of the acid-

unstable crc-amylase was about six times greater than that of the acid-stable o-amylase. Thermal

stability and pH stability are also quite different, with the acid-stable ø-amylase being more stable

at high temperatures and low pHs, retaining about 90% catalytic activity even at pH 2.2, while the

acid-unstable enzyme is inactivated completely at pH 3.0 (Minoda ef a/., 1968).

The acid-stable form of cx-amylase from A. kawachiiwas characterised, and had an

optimal pH range much lower than the acid-unstable cr-amylase (pH ranges of 2,0-6.5 and 4.5-

9.5 respectively), but the same optimal pH (pH 5.0). The acid-stable o-amylase also has a much

higher molecular weight (approximately 85 kDa compared to 50 kDa for the acid-unstable) (Sudo

et al., 1993), The production of acid-stable cx,-amylaseby A. kawachiiseems to be solid-state

culture-specific and is affected by the moisture content in solid medium, whereby increasing the

moisture content of the solid medium decreases the production of acid-stable cr-amylase

(Nagamine et a1.,2003). Productivity and specific activity of acid-stable o-amylase is low, and

does not appear to be induced by starch or malto-oligosaccharides, but commences when the

concentration of intracellular content storage glycogen begins to decrease and an inducer such

as dextrin is present. The decrease in content storage glycogen itself may not directly act on the

synthesis of acid-stable o-amylases, but the carbon starvation conditions that are required for the

decrease in glycogen may be important (Sudo ef a/., 1993). Aside from the difference in stability

at low pHs, acid-stable a-amylase activity can be distinguished from the acid-unstable a-amylase

activity by cleaving the third glycosidic bond of malto-oligosaccharides instead of the second

glycosidic bond cleaved by the acid-unstable o-amylases (Suganuma, 1997).

Woeldike (1989, SWISSPROT Accession number P56271) isolated the gene encoding

acid-stable cr-amylase from A. niger, and the deduced amino acid sequence from the cloned

DNA showed that the acid-stable cx-amylase ol A. niger was distinct from the acid-unstable o-

amylase, Amino acid identity between the two proteins was approximately 80% (Boel ef a/.,

1990), and there were extensive structural similarities between the separate domains of the acid-

unstable and acid-stable amylase. However, a rotation of the COOH-terminal domain in acid-

unstable cx-amylase was observed relative to the acid-stable enzyme. An acid-stable o-amylase

from A. kawachii, AsaA, sharesgT% aa identity with the A. niger acid-stable o-amylase between

16


aa1-479, and beyond this contains a C{erminal region rich in serine and threonine which may

function as a raw starch binding site (Kaneko ef a/,, 1996).

1 .4.2 Acid-unstable o-amylases

Cloning of the cx-amylase (often refened lo as Taka-amylase A) genes began in several

strains of A. oryzae (Gines et a\.,1989; Tada et a|.,1989; Tsukagoshi ef a/., 1989; Wirsel ef a/.,

1989), and were subsequently cloned in a similarfashion from A. nigervar. awamori(Korman ef

a/., 1990) and other fungi to identify multiple copies of cr-amylase genes. The amino acid

sequence of an o-amylase gene from A. oryzae (Toda ef a1.,1982) was used to synthesize DNA

probes to hybridise to genomic copies of the cr-amylase genes via Southern analysis. The three

cloned o-amylase genes from A. oryzae all encode a 499 aa polypeptide, whereas the two cr-

amylase genes from A. nþer encode a 499 and 498 aa protein respectively. The cloned

sequences of the a-amylase genes contain nine exons and eight introns of almost identical

sequence in identical positions. Some of the predicted polypeptide products show a few amino

acid differences from the published amino acid sequence (Toda et al., 1982) that are not

expected to alter the functional structure of the protein [ranging from 8 aa differences (Gines ef

a/., 1989) lo 12 aa differences (Tsukagoshi et a1.,1989)1. Therefore cr-amylase seems to be

encoded by a small multi-gene family, consisting of three genes in A.oryzae, amyl, amy2(taaGl

and amy3, and two genes in A. niger, amyAand amyB.

The 5' non-coding regions ol A. oryzae amyl, amy2 and amy3 genes, and A. niger amyA

and amyB genes are essentially the same, including a typical TATA box at -32 bp and a CCAAT

element al -125 bp. Also, a putative GT rich motif was observed at positions -40 bp through to -27 bp. The 3' non-coding region of the three A. oryzae genomic DNAs have identical sequence to

69 bp downstream from the translation termination codon, after which they differ significantly from

each other (Tsukagoshi et a1.,1989). Surprisingly, the cx-amylase genes in A. niger show 98%

DNA sequence identity to those of A. oryzae in their coding regions, as well as almost identical

intervening sequences and 5' untranslated regions.

Like A. oryzae cr-amylases , A. niger amyA encodes a mature peptide of 478 aa, while

amyB encodes a 477 aa protein after the 21 aa signal peptide has been cleaved. All identified

amylase genes contain this 21 aa hydrophobic secretory signal, and eight identical introns. amyA

and amyB are identical in DNA sequence except for the last 9 bp of coding sequence and their 3'

untranslated regions. The first 69 bp of \he amyB 3' non-coding region is identical to that of A.

oryzae 3' regions, and the point at which they all diverge is the same, The nucleotide sequence of

the ø-amylase gene of A. shirousamiiis also almost identical to that oÍ A. oryzae, including the 5'

17

Chaoter 1: lntroduction

non-coding region, with only 2 bp substitutions present in the coding regions. This surprising

degree of sequence identity suggests that there has been much evolutionary pressure to maintain

the cr-amylase gene sequence and organisation, or it may be an example of a recent horizontal

transfer of genes.

1.4.3 Regulation of a-amylases

The Taka-amylase A gene (taaG2) in A. oryzae has a high-level expression, and is

induced by both starch and maltose, and repressed by an excess of glucose (Errat ef a\.,1984).

Regulation of the amylolytic genes has been best studied using taaG2 in A. oryzae and when

introduced into A. nidulans, to identify three regulators of amylolytic gene expression; the wide-

domain regulators CreA and AnCP/AnCF, and the pathway specific activator AmyR, as detailed

below,

Analysis of the 5' promoter region of cx-amylase taaG2 in A. oryzae revealed CreA

consensus binding sites (S'-SYGGRG-3'), and potential CreA binding sites are found in promoters

of other starch induced genes, such as a-glucosidase. Kato et al. (1996) demonstrated that CreA

binds to the o-amylase promoter of taaG2 in vitro, indicating that the CreA protein is involved in

the repression of the cr-amylase genes in response to glucose.

Nuclear proteins that recognise the CCAAT element and the adjacent upstream

sequence in the promoter region of the A. oryzae cx-amylase gene were identified by Nagata ef

a/. (1993) using A. nidulans as an intermediate host. An A. pidulans CCAAT element binding

protein was identified (AnCP)that not only binds to the CCAAT element of a-amylase (taaG2)in

A. nidulans, but also binds to promoters of other genes that are unrelated to amylase which

contain this CCAAT element (Kato ef al., 1997). When this CCAAT sequence was mutated rn

vivo, AnCP binding was completely abolished, and an approximate four fold decrease of

cx-amylase expression resulted, suggesting a positive function for the CCAAT element, Even

though the amylase activity was lowered when this CCAAT element was mutated, amylase

activity was still significantly induced by starch and repressed by glucose, indicating that AnCP

function is not necessary for the starch induction of a-amylase expression.

A number of CCAAT binding proteins have been independently identified in A. nidulans

that modulate gene expression, including AnCP binding to the promoter of taaG2, AnCF (4.

nidulans CCAAT factor) binding to the amdS promoter to affect acetamidase expression (van

Heeswijck and Hynes, 1991), and PENR1, binding To acvA, aatA, and ipnA anlecling the

expression of the penicillin biosynthesis (Then Berg ef a/,, 1996; Litzka et al., 1996). These

proteins complexes are homologous to the S. cerevisiae Hap complex involved in transcriptional

18

Chapter 1: lntroduction

activation, and are minimally composed of three subunits, HapB, HapC, and HapE

(Papagiannopoulos ef a/., 1996; Kato ef a/., 1998; Steidl ef a/,, 1999), Recently, a gene encoding

a novel transcriptional activator, hapX,was isolated from A. nidulans and shown to interactwith

the Hap complex (Tanaka et al., 2002). A. oryzae also contains a protein complex, AoCP,

comprised of HapB, HapC and HapE that binds specifically to a CCAAT element in the promoter

ol taaG2 (Tanaka et a1.,2000,2001). CCAAT elements are found in the promoters of many

genes, including other amylolytic genes, such as the o-glucosidase genes ol A. nidulans and A.

oryzae, indicating that they may also be modulated by the AnCP/AnCF (Hap) complex.

amy? encodes a transcriptional activator of the starch-induced genes that was

independentlyclonedfromA. oryzaebytwoseparategroups(Petersenef a/., 1999; Gomi ef a/,,

2000). the amyR disruptant strain showed restricted growth on starch medium, and produced low

levels of amylolytic enzymes such as o-amylase and glucoamylase compared with a wild{ype

strain, indicating that AmyR is the positive regulator involved in starch/maltose induced

expression of these genes (Gomi et al., 2000), AmyR contains a Zn(ll)zCyso DNA binding

binuclear cluster in the N{erminal region (aa 28 - 54), similar to the Gal4p transcription factor

from S. cerevisiae, and a leucine zipper-like heptad motif at aa 351 which can form coiled-coil

structures, possibly acting as a homo-dimerization domain (Petersen ef a/,, 1999; Gomi ef a/.,

2000). Petersen et al. (1999) identified two different AmyR binding sites, both containing the

triplet CGG that is known to interact with a Gal4p-like Zn(ll)zCyso binuclear cluster (Reece and

Ptashne, 1993). One site, (5'-CGG-Na-CGG-3') is a conventional Gal4p{ype binding site, whilst

the second site, (5'-CGGAAATTTAAA-3') differs from most other Gal4p{ype binding sites as it

only has one CGG triplet. AmyR binding sites were identified in a number of starch-induced

promoters from both A. niger and A. oryzae (Petersen et al., 1999), and previously identified

regions required for starch induction including Region llla (Minetoki et a1.,1996) and the starch

responsive element (SRE) (Tani et a/., 2000) contain these AmyR binding sites,

Transcriptional analysis revealed that the amyR gene was expressed at the same level in

the presence of glucose as in the presence of maltose, compared to the amylolytic genes that

were only highly transcribed in the presence of maltose (Gomi et a1.,2000). Consistent with this

Petersen et al. (1999) observed that AmyR had an effect on the o-amylase promoter under both

inducing and non-inducing conditions, and hypothesised that the presence of an inducer, such as

maltose, transforms AmyR into a better activator.

Gomi ef a/. (2000) identified an amylase cluster, containing amy1, agd,A (encoding cr-

glucosidase) and amyA (encoding o-amylase) clustered within a 12 kb fragment in A. oryzae.

19

Chapter 1: lntroduction

fhe amy? and agdA genes share a 1.5 kb upstream region, and are transcribed in opposite

directions.

1.5 Aims and Objectives

ln A. nidulans three genes involved in carbon catabolite repression have previously been

characterized; creA, which encodes a transcriptional repressor protein, creB which encodes a

deubiquitinating enzyme, and creC which encodes a protein that contains five WD4O-repeat

motifs and a proline-rich region. CreB and CreC form a complex in vivo and it has been proposed

that this complex acts to stabilize the CreA repressor protein by removing ubiquitin moieties that

target CreA for destruction via the 265 proteasome or alter its function. The creD34 mutation was

identified as a suppressor of the effects of the creC27 mutation, and it was also shown to

suppress the effects of the creBlS mutation, suggesting a role for CreD in this regulatory network.

The acrB2 mutation shared some phenotypes with creD?4, but others had not been tested.

The main objective of this project was to further elucidate the de/ubiquitination network

regulating carbon catabolite repression, The first aim was to clone and characterize creD, and

determine its role in carbon catabolite repression. The gene was cloned by complementation and

sequenced, Bioinformatic analysis led to a proposed function, and a bacterial-2-hybrid sueen

was performed to identify proteins that interacted with CreD. The second aim was the

characterization of lhe acr9gene. acrB was tested forthe ability to suppress the effects of the

creB and creC mutations, indicating a role in carbon catabolite repression. Sequence analysis

and phenotypic analyses of three mutant acrB alleles were performed to define impofiant regions

of the gene.

An amylase cluster was identified and preliminary characterization begun by R. Murphy,

Here the sequence analysis of the cluster was completed and bioinformatic analysis performed. lt

was shown that a previously identified amylase in A. nidulans, amyB, did not form part of this

amylase cluster.

This work describes the cloning and analysis ol creD, and the characterization of the

acrB gene, two components of a regulatory network controlling carbon source utilization in the

filamentous fungus A. nidulans that involves ubiquitination and deubiquitination.

20

Chapter 2: Materials and methods

CHAPTER 2: MATERIALS AND METHODS

2.1 Materials

General laboratory chemicals and growth media were of analytical research grade and

were purchased from a range of commercial manufacturers.

2.1.1 Enzymes

Restriction enzymes (and corresponding 10 X buffers) were purchased from Boehringer-

Mannheim, New England Biolabs (NEB) and Promega. The remaining enzymes were obtained

from the following manufacturers:

Klenow fragment Geneworks, Adelaide, Australia #KF-1

T4 DNA ligase Boehringer-Mannheim, Germany #481220

Iaq DNA Polymerase Geneworks, Adelaide, Australia #BTQ-1

PfuTurb@ DNA Polymerase Stratagene, La Jolla, CA #600250

Lysing enzymes Sigma Chemical Co., St. Louis, MO #L-1412

All enzymes were used following the manufacturers instructions, in the appropriate reaction

buffers.

2.1.2 Radioactive isotopes and nucleic acids

[cr-szP] dATP (3000 Ci/mmol)

[o-szP] dATP (3000 Ci/mmol)

[cr-ozP] dUTP (3000 Ci/mmol)

Mixed random decamers

Ultrapure dNTP Set

Radiochemical Centre, Amersham, UK

PerkinElmer Life Sciences lnc., Boston, MA

PerkinElmer Life Sciences lnc., Boston, MA

Geneworks, Adelaide, Australia

Amersham Pharmacia Biotech lnc.,

Piscataway, NJ

#440004

#ADA-32

#ARU-321

#27-2035-02

2.1.3 Molecular weight markers

100 bp DNA ladder

1 kb DNA ladder

100 bp DNA ladder

¡, DNA restricted wilh Hindlll

New England Biolabs, Beverly, MA #323-1

New England Biolabs, Beverly, MA #323-2

Promega, Madison, Wl #G2101

Finnzymes, Finland #F301S

21


2.1.4 Bacterial strains

The strains of Escherichia coll used in this study are presented are in Table 2.1

Table 2.1 Strains of E coliused in this study,

2.1.5 A. nidulans strains

The genotypes of A. nidulans strains used in this study are presented in Table 2.2.

Tabfe 2.2 A. nidulans strains used in this study.

2,2 Legend. symbols are described in Clutterbuck (1993, 1997). FGSC = Fungal Genetics Stock CenterAll strains are veAl mutants

Strain Genotype Reference

NM522 supE L,(lac-proAB) L(mcr9-hsdSLr'f)5 F lproAB laclq IacZ

LMtaGough and

Murray (1983)

DH5cr F- mcrA LlacUl69 @80d lacZ LMl5) hsdRl7 recAl endAlgyrA96 thi-l relAl

Hanahan (1983)

B2H I

reporterL(m c r A) 1 8 3 L(m c r C B -h sd S M B - m r r) 1 7 3 e n d A 1 s u p E44

recAl gyrA96 relAl laclF laclq bla lacZKan¡1

Stratagene#2001 80

B2H II

reporterL(mcrA) 1 83 L(mcrCB-hsdSM H-m rr) 1 73 endAl his9 supE44thi-l recAl gyrA96 relAl laclF' laclq H\SS aadAKanl

Stratagene#200192

Strain Genotype Origin

wild-type biAl;ribo82 Derived from Pateman et al. (1967)

creA204 biAl creA204;niiA4 Derived from Hynes and Kelly (1977)

creA204b yAl creA204, riboB2 Derived from Hynes and Kelly (1977)

creBl937 yAl pabAl ;creBl 937;riboB2 Lockington and Kelly (2001)

creC27 biAl;creC27 Hynes and Kelly (1977)

creD34 creD?4;riboB2 Derived from Kelly and Hynes (1977)

acrB2Y yA2;acrB2;choAl FGSC869

acrB2G biAl;acrB2;riboB2 Derived lrom acrB2Y

acrBl4 pabaAl ;acrBl 4;pantoBl 00 Supplied by HN Arst

acrBl5 pabaAl;acrB|5 Supplied by HN Arst

creA204 creD34 creA204; creD34; niiA4 Derived from Kelly and Hynes (1977)

creBl5 creD34 creBl 5 creD34; riboB2 niiA4 Derived from Kelly and Hynes (1977\

creC27 creD?4 creC27 creD34;riboB2 niiA4 Derived from Kelly and Hynes (1977)

creA204 acrB2 creA204; acrB2; niiA4 This work

creBl937 acrB2 yA- pabaAl ;creBl 937 acrB2 This work

creC27 acrB2 yA2;creC27 acrB2;choAl This work

creD14 acrB2 creD34 acrB2;niiA4 This work

creC27creD34/creC27 creù

Diploid trom creC27 and creC27creD34strains (this work)

22


2.1.6 Oligonucleotides

Custom oligonucleotides used in this study (Table 2.3) were all purchased from

Geneworks (Adelaide, Australia).

Table 2.3 0ligonucleotide primers used in this study

Primer name Primer sequence (5'to 3') Use Location

APYF TTTCTGCTCGTCTTGGTC 22Notrel1 PCR of APY reqion ch3APYR AGGTGTGGCTGTGTTGTC 22Notrel1 PCR of APY reqion ch3PHF GACAACACAGCCACACCT 22Notrel1 PCR of PH reoion ch3PHR AAGCCTCAACCTCTCCAC 22Notrel1 PCR of PH reqion ch3MidF GAAACGCCAGACACAGAC 22Notrel1 PCR of Mid region ch3MidR TGGAGAAGTCGTGATTGG 22Notrel1 PCR of Mid reoion ch3STF TACTCCGACAGCAACCAT 22Notrel1 PCR of ST reqion ch3STR AGTTCGTTGGCAGTCTCA 22Notrel1 PCR of ST region ch3NovelF GCCTGAATTACCAGTTGC 22Notrel1 PCR of novel reoion ch3NovelR CGGAGTAGATTGCTGTGG 22Notrel1 PCR of novel reoion ch3DF1 GTGGAGGTGAGCAAGAAG creD PCR/sequence ch3Dt2 CAAGCTGATCCCTCTCCT creD PCR/sequence ch3DF3 GGTATCGGCATCAGAACA creD PCR/sequence ch3DF4 ACGTGCTCCTGAAACAGA creD PCR/sequence ch3DF6 GCGGAGGCGGCAGTGCAA creD PCR/sequence ch3DR1 CTATGACTTCGGGAATGG creD PCR/sequence ch3DR2 GGTTAGCGCATCCATACA creD PCR/sequence ch3DR3 ATACTTGCGGCCTATTTC creD PCR/sequence ch3DR4 ATGAGCACCAACAGAAAC creD PCR/sequence ch3DR5 GTACGCGAGAACTGAAAC creD PCR/sequence ch3BThulAFRl ACGAATTCAC GACCTAATCTCAGT hulAB2H PCR/sequence ch3BThulARBam AAGGATCCTGACCGGAAGTAGATGAG hulAB2H PCR/sequence ch3TRGcreDFBam ATGGATCCCGTGGAGGTGAGCAAGAAG creD BZH PCR/sequence ch3TRGcreDRXho TATCTCGAGCTATGACTTCGGGAATGG creDB2H PCR/sequence ch3TRGapvAFRI ATGAATTCCTG G GAAG ATCAAGATG apvABZH PCRisequence ch3TRGapvARXho MTCTCGAGTCMGCCACTAATCCTG apvAB2H PCR/sequence ch3huIASEQF GAGCGGAGAGCGCACCAG sequence oBThulA ch3huIASEQR GCTGTTGCTGGATGGTGG sequence pBThulA ch3apyACF CAAACCGCAATCTGAAGC PCR for cloning apyA ch3apyAOR TGCCTAAACTGCCTCAAG PCR for cloning apyA ch3

KBB GCGAAGGGAAATGCAGAC acr9PCR1sequence ch4KB ATGGGCTAAATCGAGAGC acrBPCR| sequence ch4AK GCAATGGATGGTTTCTGC acr9PCR| sequence ch4SF CTCGCCTCGCCAATAGAT acr?PCR| sequence ch4KF CGCGTGGATACAGAAGCG acrB PCRI sequence ch4AS CAATCTCTGCCTTGGCGG acrBPCR| sequence ch4SB AAGAGGCTACCGCCGCTC acrB PCRI sequence ch4SBB GCGAGCGGTGGCTTGGCA acr9PCR1 sequence ch4KBF GACACGCCACCATTCGTC acrB PCRI sequence ch4SBF CTTGGGCACATTGGCGTC acrB PCRI sequence ch4acrBl AGTCTTGGCTGCTATCCG acrB PCRI sequence ch4acr82 TCCTTATGCGCGGCAGTG acrB PCRI sequence ch4acrBS GGGCAACGGTGGTCATCA acrB PCRI sequence ch4acrB4 TCTTCCTAACCGTACCCG acrB sequence ch4

23


Table 2.3 Legend FSP (Forward Sequence Primer), RSP (Reverse Sequence Primer), f7,T7 and SP6 are all

commercially designed primers, and were used for sequencing inserls in standard cloning vectors such as

pBluescript and pGEMTeasy. pTRGforward and pTRGreverse are commercially designed primers for sequencinginserts of pTRG constructs in the BacterioMatch bacterial-2-hybrid system.

Primer Name Primer sequence (5'to 3') Use Location

acrBS AACCAGTCACTACACGGA acrB PCRI sequence ch4acrB6 GATCCAG CAGTACCGTTGTATGTT nested 5' RACE ch4acrBTSaiRl CGAATTCACCTCCGCAACCCTCA acr9BZH PCR/sequence ch4acrBT3BAM2 GTGGATCCTAGTTGGCTGGCTGGC acrBB2H PCR/sequence ch4acrTRGSaiBAM CTGGATCCTCTCCGCCACCCTCAT acrBBZH PCR/sequence ch4acrTRG3Rl TGGAATTCAGTTGGCTGGCTGGC acrBB2H PCR/sequence ch4

AMYOl CAGCCAGCACTGTCGCC amvlase cluster sequence ch5AMYO2 TACGGCATTCCGATGTT amvlase cluster sequence ch5AMYO3 AAGATCATGGCCCGTCT amvlase cluster sequence ch5AMYO4 TCTTTCCACTCGCCTTCG amvlase cluster sequence ch5AMYOS CACAGAACGCCCTAACCC amvlase cluster sequence ch5AMYO6 CGGCGGACTCGGCAACTG amvlase cluster seouence ch5AMYOT CCGTCTGTCCTGAGTACA amvlase cluster sequence ch5AMYOs TGACCTCTGCTACAGTCG amylase cluster sequence ch5AMYO9 AGATCTTGGCCCGTCTGG amvlase cluster sequence ch5AMYOlO TCCTGGCGTCGTGGTTGG amvlase cluster sequence ch5AMYO11 GGCAGAATGAGACCTACC amylase cluster sequence,

amvAPCR for riboorobe

ch5

AMYOl2 CATGGGATAGTTCAGCAC amvlase cluster sequence ch5AMYO13 GGTAGGTCTCATTCTGCC amvlase cluster sequence ch5AMYOl4 CGCCCACCTGCGTGAAGC amvlase cluster sequence ch5AMYO15 CTAGAATCCTCGGTAGCG amvlase cluster sequence ch5AMYO16 GGGAAGAGGTACGAGTGG amvlase cluster seouence ch5AMYO17 ACCTCCGCTTACGCTTCC amvlase cluster sequence ch5AMYO18 CGAGCAGTTCTTCACCGG amylase cluster sequence

amv?PCR for riboprobe

ch5

AMYO19 GCCTCACCAGCCGCTCCG amylase cluster sequence

amv?PCRfor riboprobe

ch5

AMYO2O CTTGGTACATCTATCGCC amvlase cluster sequence ch5AMYO21 CATCTACGGACAGCACGC amvlase cluster sequence ch5AMYO22 TGCCGAGTACTGCTGACG amvlase cluster sequence ch5AMYO23 GAAGAGTCTTAACGTCCC amvlase cluster seouence ch5AMYO24 GGTACGACTACAGGCCGC amvAPCR for riboprobe ch5

AMYS ATCCAGTACTAACGCCGC amv9PCR for riboprobe ch5AMYs CCGTATGCGGTCTGCACC amvBPCR for riboprobe ch5

pTRGforward CAGCCTGAAGTGAAAGAA sequence pTRG constructs Chs 3,4pTRGreverse ATTCGTCGCCCGCCATAA sequence pTRG constructs Chs 3,4

FSP GTAAAACGACGGCCAGT

HSP AACAGCTATGACCATG

17 AATACGACTCACTATAG

T3 ATTAACCCTCACTAAAG

SP6 GATTTAGGTGACACTATAG

24


2.1.7 Vectors

Vectors used in this project are presented in Table 2,4

Table 2.4 Plasmids used in this study.

Plasmid Description Manufacturer

pPL3 riboBr in pUC19, used as selectable marker

for cotransforming A. nidulans

Oakley et al. (1987)

pBluescript ll SK+ General cloning vector Stratagene

pGEM-Teasy Cloning of PCR products Promega

PBT Bait vector of BacterioMatch 82H system Stratagene

pTRG Target vector of BacterioMatch B2H system Stratagene

2.1.8 A. nidulans DNA libraries

The A. nidulans gDNA BacterialArtificial Chromosome (BAC) library used was made by

Dr. Ralph Dean, Director, Center for lntegrated Fungal Research, Norlh Carolina State University

(for reference see Zhu et a\.,1997) and kindly provided by Dr. A. Andrianopoulos, Department of

Genetics, University of Melbourne. The A. nidulans chromosome ll specific gDNA cosmid library

is described in Brody ef at (1991).

The A. nidulans cDNA library (kindly provided by S. Osmani) was constructed from

pooled RNA isolated from a range of developmental stages of mycelia grown in glucose

conditions, and was used for determination of intron positions and as the basis for the

construction of the bacterial-2-hybrid (B2H) cDNA library (R. Lockington, unpubl. data).

2.1.9 Kits and miscellaneous mater¡als

BacterioMatchTM Two-hybrid SystemVector Kit

DNeasp Plant Mini Kit

First ChoiceTM RLM-RACE Kit

Hybond-N+ nylon membrane

Nucleospino RNA Plant Kit

PSlYClone BAC DNA kit

QlAquick PCR Purification Kit

Riboprobeo Combination System -T3/T7 Kir

Stratagene, La Jolla, CA

Qiagen, Valencia, CA

Ambion lnc., Austin, TX

Amersham, UK

Machery-Nagel, Germany

Princeton Separations, Adelphia, NJ

Qiagen, Valencia, CA

Promega, Madison,Wl

#240065

#691 04

#1 700

#RPN3O3B

#740949

#PP120

#28104

#P1450

25

2.2 Solutions, butfers, and media

2.2.1 Solutions and buffers

All solutions and buffers were prepared using millipore water and where appropriate,

were autoclaved. Solutions not able to be autoclaved were sterilised by filtrations through a 0,2

pm filter. Solutions and all other buffers routinely used in this study were as follows:


RPA lll Ribonuclease ProtectionAssay Kit

Wizardo P/us SV Minipreps DNApurification systems Kit

X-ray film

Blotto

100 X Denhardts solution

10 X Loadino dve

10 X MOPS buffer

10 x 0liqolabellinq buffer

Phenol/chloroform

Prehvbridisation(formamide ) buffer

Prehvbridisation(phosphate) buffer

RNA loadinq buffer (M0PS)

l XTE

Ambion lnc., Austin, TX #1415

Promega, Madison, Wl, #41460

Fuji RX MedicalX-ray Film, Fuji

Photo Film Co., Ltd., Tokyo, Japan

#03Ê220

10% skim milk powder, 2 mg/mlsodium azide

2% (wlv) Ficoll 400, 2% (wlv) polyvinylpyrrolidone, 2% (w/v) BSA

0.25% bromophenol blue, 0,25% xylene cyanol FF, 25% Ficoll 400

200 mM MOPS, 50 mM anhydrous NaAc, 10 mM EDTA, adjustedto pH 7.0

0.5 M Tris-HCl pH 6.9, 0.1 M MgSO¿, 1 mM DTT, and 0.6 mM

each of dCTP, dGTP, and dTTP

50% (v/v) phenol, 48% (vlv) chloroform, 2% (vlv) isoamyl alcohol,

buffered with an equal volume of Tris-HCl (pH 8.0), 0,2% (v/v) B-mercaptoethanol

40% formamide (v/v), 4 x SSPE, 1% SDS, 5% blotto, 100 pgsonicated salmon sperm DNA

10 mM sodium orthophosphate, 5 X SSC,2% blotto,0.4% SDS,

100 pg sonicated salmon sperm DNA

50% (v/v) formamide, 37% (vlv) formaldehyde, 1 x MOPS buffer, 1

x loading dye, 10 mg/ml ethidium bromide

50% (v/v) formamide, 12% (vlv) formaldehyde, 10 mM sodiumorthophosphate, 1 x loading dye, 10 mg/mlethidium bromide

0.1 5 M NaCl, 0,01 5 M NasCoHsOt.2HzO, pH 7.2

0.18 M NaCl, 10 mM NaHzPO+, 1 mM EDTA, pH7.4

40 mM Tris base, 20 mM NaAc, 2 mM EDTA, pH 7.8 with glacial

acetic acid

10 mM Tris-HCl(pH 8.0), 1 mM EDTA

26


2.2.2tlledia

Aspergillus complete and minimal media were as described by Cove (1966). Carbon

sources were added at a final concentration of 1% (w/v) unless stated othenruise, Nitrogen

sources were added to a finalconcentration of 1OmM unless othen¡rise stated.

Standard bacterial media (L-broth, Luria Bertani Agar etc.) were prepared as described in

Sambrook et al. (1989). Where required, ampicillin or chloramphenicol were added to a final

concentration of 50 pg/mlor 170 ¡rg/ml respectively, unless othenryise stated.

2.3 Methods

Standard molecular techniques were performed as outlined in Sambrook et al. (1989).

2.3.1 Manipulation oÍ A. nidulans

Growth testing and meiotic analyses were pedormed using the methods described by

Cove (1966). Genetic manipulations were carried out using techniques described by Clutterbuck

(1974). Transformation experiments were carried out using the method of Tilburn etal, (1983),

typically adding 100 mg Lysing Enzymes (Sigma Chemical Co.)for protoplasting. Transformants

from co-transformation experiments were selected using lhe riboB+ selectable marker plasmid

pPL3 on media lacking riboflavin. The genotypes of all double mutant strains constructed during

this project were verified by molecular analyses.

2.3.2 Nucleic acid isolation

A. nidulans genomic DNA was isolated using the DNeasf Plant Mini Kit according to the

manufacturers instructions. Prior to the purchase of this kit, gDNA was isolated from wet

mycelium by the method of Lee and Taylor (1990).

BAC DNA was isolated from E coli using the PSlYClone BAC DNA kit according to the

manuf acturers instructions.

Plasmids were isolated from E. coli using the Wizardo P/us SV Minipreps DNA

purification systems Kit according to the manufacturers instructions.

Cosmids and fosmids were isolated from E. coliusing the Wizardo P/us SV Minipreps

DNA purification systems Kit according to the manufacturers instructions, except for the final

elution step which was performed with 50 ¡rl of nuclease{ree water pre-warmed to 65"C and

incubated at room temperature for 5 minutes before the final centrifugation.

Total RNA was isolated from A. nidulans mycelia grown in specified conditions, using the

Nucleospino RNA Plant Kit according to the manufacturers instructions.

27


2.3.3 Polymerase Chain Reaction (PCR)

Optimal conditions for amplification of DNA fragments via the Polymerase Chain

Reaction (PCR)were determined for each set of template and primers. Generally, these reactions

were as outlined in lnnis and Gelfand (1990), with 100 ng of double-stranded DNA and 100 ng of

each specific oligonucleotide primer in a final reaction volume of 50 pl. DNA was typically

amplified in a programmable PTC-200 DNA Engine (MJ Research, lnc.) as follows: an initial

denaturation at 95"C for two minutes, followed by 25-30 cycles using these conditions:

denaturation at 95"C for one minute, a 30 second annealing step and extension al72"C for one

minute. The annealing temperature was modified to suit specific templateiprimers sets and

ranged between 48"C and 60"C. The extension time also varied depending on the size of the

fragment to be amplified, allowing a 1 kb/min synthesis rate.

2.3.4 DNA sequencing

PCR products were purified using the QlAquick PCR Purification Kit prior to sequencing,

and plasmid DNA required no further purification after isolation as describe above. Sequencing

reactions were performed using a Prism Big Dye Terminator Kit (Applied Biosystems-Perkin

Elmer) as outlined in the manufacturers instructions. Dye terminator reactions were

electrophoresed on an ABI Prism Model 377 automated sequencing machine at the IMVS

sequencing facility (lnstitute of Medical and Veterinary Science, Frome Rd, Adelaide SA 5005).

2.3.5 Rapid amplification of 5'cDNA ends (5'-RACE)

5'-RACE was peÍormed on total RNA from D-glucose-grown mycelium using the First

ChoiceTM RLM-RACE Kit (Ambion), following the manufacturers instructions.

2.3.6 DNA gel electrophoresis and Southern transfer

DNA for Southern blot analysis was separated by gel electrophoresis through 1%

agarose in 1 x TAE buffer, depurinated by treatment with 0.25 M HCI for 10 minutes and

transferred to Hybond N+ membrane (Amersham) by alkaline transfer in 0.4 M NaOH for four

hours or 20 x SSC overnight, as recommended by the manufacturer. Following overnight transfer

in 20 X SSC, filters were treated with 0.4 M NaOH for 20 minutes. DNA filters were prehybridised

for at least 90 minutes aI42"C in prehybridisation (formamide) buffer. Denatured labelled DNA

probe was added directly to the prehybridisation mix and incubated al 42"C overnight, Filters

were washed with 2 x SSC, 0,1% SDS for at least 15 minutes aT 42"C, and then 0.5 x SSC, 0.1

% SDS lor 20 minutes al 42"C and then exposed to X-ray film or to a phospho-imager plate.

28


Sometimes an additional 10 minute high stringency wash with 0.1 x SSC,0.1% SDS was

required. Membranes were stripped of probe by placing the membrane in a 400 ml solution of

boiling hot 0,5% SDS for 10 minutes, then rinsed in 2 X SSC and stored in plastic wrap at 4"C.

2.3.7 RNA gel electrophoresis and Northern transfer

Two methods of RNA gel electrophoresis were used during the course of this project.

Using the "MOPS' method, 1 pg - 5 pg total RNA was electrophoresed through 1.2% agarose -

0.6 M formaldehyde gels in 1 x MOPS running buffer, RNA samples were added to RNA loading

buffer (MOPS) and heated to 6B"C for 15 minutes prior to loading. Alternatively, using the

"phosphate" method 1 trg - 5 pg total RNA was electrophoresed in 1.5% agarose - 0.6M

formaldehyde - 10 mM sodium ofihophosphate gels in 10 mM sodium orthophosphate running

buffer. RNA samples were added to RNA loading buffer (phosphate) and heated to 68"C for 15

minutes prior to loading.

RNA was transferred to Hybond N+ (Amersham) by alkaline transfer in 0.04 M NaOH for

2-3 hours. Denatured radioactively labelled probe (DNA or RNA) was added directly to the

prehybridisation mix and incubated overnight. Filters were washed with 2 x SSC,0.1% SDS for at

least 15 minutes, and then 0.5 x SSC, 0.1 % SDS for 20 minutes and then autoradiographed.

Sometimes an additional 10 minute wash with 0.1 x SSC, 0.1% SDS was required. Two

hybridisation conditions were used on the Northern fifters. Hybridisations and washes were all

performed al 42"C when prehybridisation (formamide) buffer was used, and at 65"C when

prehybridisation (phosphate) buffer was used.

2.3.8 Ribonuclease Protection Assays

Ribonuclease Protection Assays were typically pedormed on 5 ¡rg total RNA using the

RPA lll Ribonuclease Protection Assay Kit, according to the manufacturers instructions.

2.3.9 Radioactive labelling of probes

For DNA probes, 50-200 ng DNA was radioactively labelled with [cr-szp] dATP by primer

extension of random decamer oligonucleotides using the method of Hodgson and Fisk (1987).

Radiolabelling by PCR was also performed on PCR templates as per the standard PCR protocol,

except that the dNTPs are replaced by a mixture of dCTP, dGTP and dTTP, and [o(-32P] dATP,

and typically the cycle number was reduced to I cycles.

For RNA probes,200-1000 ng RNA was radioactively labelled with [cx-ezP] dUTP using

the Riboprobeo Combination System -fgn7 Kit according to the manufacturers instructions.

29


2.3.1 0 Bacterial-2-hybrid system

Protein-protein interactions were investigated utilising Stratagene's BacterioMatcho I

Two-Hybrid System Vector Kit (B2H). B2H I reporter cells supplied with the kit were made

competent by the CaClz method, and transformed with relevant constructs and incubated

overnight at 30"C (Sambrook et a/., 1989). lnteractions were determined to be positive as

measured by growth on LB-agar plates supplemented with 750 pgiml carbenicillin, 15 pg/ml

tetracycline, 34 pg/ml chloramphenicol, and 50 pg/ml kanamycin, and validated by a blue colour

reaction on X-gal indicator plates grown overnight at 37"C (LB-agar plates supplemented with

40pg/ml X-gal [5-bromo-4-chloro-3-indolyl-B-D-galactoside] and 20pg/ml IPTG [isopropyl-B-D-

thiogalactopyranosidel as a competitive inhibitor).

The BacterioMatcho ll Validation reporter cells became available in late 2003 and are

compatible with constructs made for use with the B2H I reporter cells. The B2H ll reporter cells

were made competent by the Ca0lz method, and transformed with relevant constructs and

incubated overnight at 37"C (Sambrook et a/., 1989). lnteractions were determined to be positive

as measured by growth on "Selective Screening Medium" consisting of minimal media plus 5 mM

3-amino-1 ,2,4-triazole (3-AT), 25 ¡qlnl chloramphenicol and 12,5 pg/ml tetracyline, and

validated by growth on "Dual Selective Screening Medium" consisting of minimal medium plus 5

mM 3-AT and 12.5 pg/ml streptomycin,25 pg/ml chloramphenicol and 12.5 ¡rg/ml tetracyline,

with all media prepared as outlined by the manufacturer. The pBTLGF2 and pTRGGALIIp

constructs provided with the B2H kit served as a positive control.

2.3.11 Amylase secretion plate tests

Culture media (100 ml) were inoculated with conidia (from either wild{ype or creA204b

mutant strains) and grown in conditions as described below:

16 hrs at 37"C in medium containing either 1% D-glucose (v/v),0.1% D{ructose (v/v),

1% starch (vlv),1% maltose (vlv),1% D-glucose (viv) and 1% starch (viv), or 1%

D-glucose and (viv) 1% maltose (v/v)

16 hrs at 37"C in medium containing 0.1% D{ructose (v/v), then either addition of 1%

starch (v/v) or 1% maltose (v/v) and reincubation for 4 hrs at 37"C

16 hrs at 37"C in medium containing 1% glucose (v/v), then addition of 1% starch (viv)

and reincubation for 4 hrs at 37"C

Prior to harvesting the mycelia, 20 ¡tl of the liquid culture medium (containing any

secreted cr-amylase) was plated onto 0.5% starch plates, and incubated at 37"C for t hr, 4 hrs or

16 hrs. Then the starch plates were flooded with iodine, which stains starch blue, and the amount

30


of cr-amylase activity was deduced from the amount of clearing seen on the starch plate, where

the starch has been broken down by ø-amylase.

The harvested mycelia from these overnight cultures were used to isolate RNA using

methods described above.

2.3.12 Sequence analyses

DNA sequences obtained from automated sequencing reactions were visualised using

the Chromas V2.23 (Technelysium) programme. Sequence analyses were performed using

programmes accessed via the Australian National Genomic lnformation Service (ANGlS,

http://wwwl,anqis.oro,au/pbinMebANGlS/wrapper.pl) facility or NEBcutter Version 2

(http://tools.neb.com/NEBcutter2/index.php). Database searches were perlormed via the National

Center for Biotechnology lnformation (NCBI; http://www.ncbi.nlm.nih.qov). The A. nidulans

genome sequence was accessed via the Whitehead lnstitute's Center for Genome Research

website (

2.3.13 Phylogenetic analyses

Phylogenetic and molecular evolutionary analyses were conducted using MEGA version

2.1 (Kumar et al., 2001). Sequences used for analyses were identified as proteins containing

arrestin domains via a BlastX (Altschul et a|.,1990) search using creD, initially against the NCBI

database (http://www.ncbi.nlm,nih.qov/blasVBlast.cqi), and then against individual organism

genome sequence databases with results as follows: A. fumigatis AfApyl (AFGDP80TR.69)

obtained from The lnstitute for Genomic Research (TIGR) A. fumigatis genome database

(http:/iwww.tiff.ors/tdb/e2k1lafu1/), N. crassa NcrApyl (E4A28290.1) obtained from the N.

crassa genome database (http://www-qenome.wi.mit.edu/annotations/f unqi/neurospora4, S.

pombe SpomApyl (SPCC5B4.15C) and SpomApy2 (SPAC31A2.12) obtained from the S, pombe

genome database (htto://www.qenedb,orqifenedb/pombeiindex.isp), and S. cerevisiae ScRodlp

(YORO18W) and ScRog3p (YFR022W) obtained from the S. cerevisiae genome database

(http://veastoenome.orq/). The alignments of amino acid sequences were generated using the

ClustalW programme (Thompson et al., 1994) accessed via ANGIS, Four regions of high

sequence conservation within the arrestin domains were used for phylogenetic analysis,

corresponding to aa 41 to 68, 107 to 162, 181 lo 226 and 282 to 331 of CreD, Phylogenetic trees

were constructed using the maximum parsimony method (Fitch, 1971)with the close-neighbor

interchange search (Nei and Kumar, 2000) or using the neighbor-joining method (Saitou and Nei,

1987), and bootstrap analysis was pedormed with 1000 replicates.

31


2.4 Nucleotide sequence accession numbers.

The sequence of the creD gene has been deposited in GenBank under Accession no,

4Y458430, The sequence of the apyA oDNA has been deposited in GenBank under Accession

no. 4Y460113. The sequence of the acrB gene has been deposited in GenBank under Accession

no, 4F485329. The sequence of the amylase cluster, including amy4, agdA and amyA, has been

deposited in GenBank underAccession no. AF208225.

32

Chapter 3: Clonino and molecular analvsis of creD

CHAPTER 3: CLONING AND MOLECULAR ANALYSIS OF ueD

3.1 lntroduction

ln A. nidulans creB encodes a deubiquitinating enzyme that forms a complex that

includes the WD4O-motif containing protein encoded by creC, and mutations in these genes lead

to altered carbon source utilization. fhe creD34 mutation suppresses the phenotypic effects of

mutations in creC and creB, and thus creD is likely to be involved in the ubiquitination aspect of

the regulatory network that involves the CreBiCreC deubiquitination complex. To further dissect

this network lhe creD gene was cloned and characterized.

Strains containing lhe creD14 mutation are resistant to the presence of acriflavine in

complete media in comparison to wild{ype strains, which are sensitive. The genetic distance

between creD and creC has been previously mapped at between 3-6 cM (Kelly & Hynes, 1977),

and creC has been located within the chromosome ll specific cosmid library (Brody ef a/., 1991)

to cosmid P24E11 (Todd et a1.,2000\. Therefore, the initial approach taken to clone creD was to

walk in the cosmid library, and test adjacent cosmids for complementation of the creD34

phenotype on acriflavine.

According to the published chromosome ll cosmid library map, cosmid 121808 overlaps

lhe creC containing cosmid P24El1. ln a preliminary study L21B0B apparently complemented the

creD34 mutant phenotype on acriflavine (R. Todd, pers. comm.), and this closeness to creC is in

keeping with the expected distance of about 30 kb between creCand creD according to previous

comparisons of physical and genetic maps. Thus the cosmid 121808 provided the starting point

for the identification of creD.

3.2 Cloning ueD via complementat¡on of the creD34 phenotype on acr¡flavine

using cosm¡ds and cosmid derivatives

3.2.1 The creD34 allele is recessive to the wild-type allele

ln order for the strategy of cloning creD by complementation to be successful it was

necessary to confirm that the creDl4 allele was recessive to the creù allele for the phenotype

observed on medium containing acriflavine. The creC27 mutant strain is sensitive to the presence

of acriflavine in complete medium, whilst the creC27creD34 double mutant strain is resistant to

the presence of acriflavine in complete medium (Boase et al., 2003). fhe creD34 phenotype has

previously been shown to be recessive to the wild-type allele on a range of media (Kelly and

33

Chapter 3: Cloninq and molecular analvsis of creD

Hynes, 1977), but the dominance properties on acriflavine containing medium was not tested. ln

order to confirm that the creD74 mutation was recessive to the wild-type allele for its phenotype

on acriflavine a creC27creD?4/creC27creD+ diploid was constructed and the phenotype was

tested (Figure 3.1). The diploid was as sensitive as creC27 on acriflavine containing medium.

Thus the affect of lhe creD?4 mutation is recessive to the wild{ype allele on medium containing

acriflavine, allowing a complementation approach to clone creD.

3.2.2 Testing cosm¡ds 121808, P24811, and relevant derivatives for

complementation of the creD34 mutant phenotype

Cosmid 121808 was retested and parlial complementation of lhe creD34 phenotype on

acriflavine was confirmed. To narrow down the complementing region, 121808 was digested with

the restriction enzyme Sa4 and religated to excise an approximate 10 kb Sa//fragment. This

subclone, L2-Sal, containing approximately 22kb of genomic DNA insert (Figure 3.2), was used

to co{ransform a creD74; riboB2 strain together with lhe riboB+ containing plasmid pPL3. L2-Sal

complemented the creD34 mutant phenotype on medium containing acriflavine, but the sensitivity

to acriflavine was not restored completely to the level of wild-type (Figure 3.3).

Preliminary Southern analysis indicated that the cosmids 121808 and P24811

overlapped each other (R. Todd, pers. comm.), and the order of known genes glnA and creC in

P24E11 allowed orientation of the cosmid (Figure 3.2), such that a previously constructed

plasmid, p8A (R Sladic, unpubl. dafa), containing a 12 kb genomic DNA fragment (Figure 3.2)

was then used to test for complementation of lhe creDl4 phenotype. p8A partially complemented

lhe creD34 phenotype on acriflavine, and so previously constructed derivatives of this plasmid,

pX1 (10 kb gDNA insert) and pB3 (5 kb gDNA inserl), were tested and also shown to parlially

complement lhe creD34 mutant phenotype on acriflavine containing medium (Figures 3.2 and

3,3). The pB3 complementing region was refined further by digesting pB3 with Eco?land the five

resulting Eco?lfragments (designated a,b,c,d and e) of pB3 were subcloned into the pBluescript

ll SK+ vector individually and in various combinations (Figure 3.2). Additional subclones of the

pB3 plasmid were constructed by subcloning a Bglll - Clallragnent (pBb'ecd) and an EcoRl -EcoRVfragment (pBabdc') (Figure 3,2), All plasmids lacking the 'pBd' EcoRlfragment of pB3 did

not show complementation of lhe creD34 mutant phenotype on acriflavine, whilst all plasmids

containing the 'pBd' Eco?lfragment of pB3 showed complementation (Figures 3.2 and 3,3).

34

Chapter 3: Cloninq and molecular analysis of creD

D

creC27 creD+ creC27 creD34

creC2| creD}4/ creìZ7 crel+

.¡ cre}+ creD+ creC+ creD34

Figure 3.1 The creDï4 allele is recessive to the wild-type allele, All strains

were grown at 37'C on either (A) 1% complete medium for 2 days (B) 170

complete medium plus 0.001% acriflavine for 2 days or (C) 1% glucose plus 10

mM pyrrolidinone medium for 3 days. The relevant genotypes are indicated by

the key (D). For full genotypes see Materials and Methods, Table 2,2'

35

Chaoter 3: Clonino and ar analvsis of creD

IITT

Complementing

transformants

L21B0B 31120

5t120ëEOr()L2-Sal

P24811

p8A

px1

p93

ND

5/60

2t30

11t120

EcoRl EcoRl Clal EcoRl EcoRl EcoRV EcoRl Bglll

pBb 0t40

0t40

7t132

7t40

3/59

9/60

pBc

Bb'ecd

pBabdc'

pBabd

pBd

Figure 3.2 Partial map of cosm¡ds L21808, P24E11and derivatives tested forcomplementation of the creDï4 phenotype on acriflavine. Cosmids are indicatedby bold lines, and dashed lines indicate reg¡ons thought to overlap. The number ofcomplementing transformants compared to the total number oÍ riboB* transformantsis indicated in the right hand column. Double diagonal lines indicate cosmid not toscale. The regions of pB3 subcloned into pBluescript are indicated below theexpanded pB3. ND indicates that no transformation experiment was performed withthis construct,

aÐ <-b <d> +c.(---

36

Chaoter 3: Clonino and moleeular analysis of creD

D

tL2lB08 tL2-Sal

tpB3 tpBb tpBd

creDl4 wild-type

Figure 3.3 Partial complementation of the creD34 mutant phenotypo. Allstrains

were grown at 37"C on either (A) 1% complete medium for 2 days (B) 1% complete

medium plus 0.001% acriflavine for 2 days or (C) 1% glucose plus 10 mM

pyrrolidinone medium for 3 days. The strains are indicated by the key (D). For full

genotypes see Materials and Methods, Table 2.2. Transformants of the creD34

strain are designated tX, where X represents the transforming DNA. Each

transformant shown is representative of a number of similar transformants generated

in the same transformation experiment.

37


3.2,3 Sequence analysis of pB3

The five EcoRlfragments of pB3 were sequenced using standard forward and reverse

sequence primers and specific oligonucleotides were designed to generate the remaining

sequencing after assembly of these five regions, This analysis revealed the presence of two

hypothetical genes within the 5 kb plasmid pB3 (Figure 3.4, Appendix A), largely contained within

either the 2349 bp'pBb' or 1651 bp 'pBc' subcloned EcoR/ fragments,

The pBb region contains a hypothetical gene that encodes a protein homologous to the

hypothetical protein Gonlp from S. cerevisiae, as determined by BlastX analysis, and shares

49.9Y0 aa identity with Gon1p. The GONI-like gene encodes a putative protein that contains an

ATP/GTP binding domain and a calcium-binding domain, as determined by protein motif analysis

in GCG (accessed via ANGIS), and only a short 33 bp 5' untranslated region for this protein is

found within the pBb clone, with the promoter region mainly residing in the pBd region. BlastX

analysis of the pBc sequence revealed that this subclone contains the majority of a gene

encoding a protein similar to the hypothetical protein Ysc84p from S. cerevisiae, sharing 52.3o/o

amino acid identity, and containing an SH3 domain, The start codon of the YSCB4-like gene

resides 13 bp beyond the pBc region, in the pBd fragment. Prior to the results of the

complementation analysis, the pBb and pBc regions were amplified via PCR from wild-type and

creD34 mutant derived gDNA and sequenced (Appendix A, Table A.1), There were no sequence

differences found between the wild{ype and creD34 alleles within either the pBb or pBc regions,

which is consistent with the subsequent lack of complementation of lhe creD34 mutant phenotype

seen for these subclones.

The pBd intergenic region between the GONI-like and YSCS4like genes partially

complemented the creD34 mutant phenotype on acriflavine medium, suggesting that the creD34

mutant phenotype may be the result of a promoter mutation for either or both of these genes,

Thus the 'd' EcoRlfragment of pB3 was sequenced from the creD34 mutant strain in order to

identify the molecular nature of the creD34 mutation, The pBd region was amplified via PCR from

gDNA of both a wild-type and creD34 mutant strain and sequenced (Appendix A, Table A.1). No

mutation was present within pBd, hence neither the GONl-like or YSCS4like gene encode CreD.

Thus it is likely that pBd contains a sequence that acts as a suppressor of the phenotypic effects

of the creD34 mutation on acriflavine, rather than creD itself.

The pBd region is 701 bp in length and contains several small open reading frames

(Figure 3.4). ln multiple copies this region can suppress the creD34 mutant phenotype on

medium containing acriflavine, perhaps due to the titration of regulatory proteins. The four open

reading frames of pBd larger than 20 amino acids in length were analysed by BlastX against the

38

Chaoter 3: Clonino and analvsis ol creD

p83

<- a-) <_b <- d-> ¡ <_ c__________Ð ¡ <- s-> I

EcoRl EcoRl EcoRl Bgill

t- like gene ke gene

pBd

49 aa 73 aa.(-

46 aa t42 aa

Figure 3.4 Map of the hypothetical genes conta¡ned within pB3, and ORF of theintergenic region pBd. The five regions of pB3, a, b ,c ,d, and e, are indicated by

the double headed arrows corresponding to their Eco4l boundaries. The block

arrows indicate the two hypothetical genes located within this region encoding the

Gonlp homologue, and the Ysc84p homologue, The 701bp region of pBd that

partially complemented the creD34 mutant phenotype is expanded, and the slim

arrows indicate ORF contained within this region.

39


NCBI database, and no highly similar sequences were identified, and thus they are unlikely to

encode a protein.

Since regions of DNA (such as pBd and L2-Sal) could suppress the creD14 mutant

phenotype of resistance to acriflavine in complete medium, transformants that appeared resistant

to the presence of acriflavine were tested on a wider range of media. A creC27creD34 double

mutant strain was transformed with the pBd suppressor plasmid, and the phenotype was tested

both on medium containing acriflavine and medium containing allyl alcohol, as the creC27 slrain

is sensitive to the presence of allyl alcohol and the creC27creD34 double mutant strain is

resistant to the presence of allyl alcohol. As expected, the pBd plasmid again suppressed the

phenotype ol creDl4 on acriflavinein a creC2Zbackground, but did not complement the creD74

mutant phenotype on medium containing allyl alcohol, indicating that this suppression of the

creD34 mutant phenotype is specific for acriflavine. Arst et al. (1982) showed that lhe creD34

mutation led to weaker growth than wild-type on medium containing 10 mM pyrrolidinone as a

sole nitrogen source. fhe creD34 phenotype on pyrrolidinone as a sole nitrogen source was

shown to be recessive in lhe creC27creD?4/creC2TcreD diploid (Figure 3.1C), allowing this

phenotype to be used in conjunction with the phenotype on acriflavine to test for

complementation. None of the transformants that complemented the creD34 mutation on

acriflavine containing medium showed complementation for growth on medium containing 10 mM

pyrrolidinone as a sole nitrogen source (Figure 3.3C), again indicating that a suppressor of the

creD?4 phenotype on media containing acriflavine alone had been successfully cloned, but was

of no further interest.

3.3 Complete complementation of the creD34 phenotype

As the transformants that appeared to complement the creD34 phenotype on medium

containing acriflavine failed to complement on medium containing pynolidinone as a sole nitrogen

source, the P24E11 and 121808 cosmid were tested no further. Other cosmids that were also

adjacent to P24E11 according to the published cosmid map were tested for complementation

(23807,22811,22A10,23E03 and 21G07) but none complemented the creD34 phenotype on

medium containing pyrrolidinone as a sole nitrogen source. Since these cosmids should span the

3-6 map units between creC and creD, the ordering of the cosmid library in this region was

checked, Allcosmids used thus far in transformation experiments, including P24811 and 121808,

were digested with Sac/| transferred to a nylon membrane and probed with oligolabelled pB3.

The only cosmid containing pB3 was P24E11, the cosmid from which pB3 was derived. Therefore

the cosmid library map was not in order in this particular region. To identify cosmids that were

40

Chaoter 3: Cloninq and molecular analysis of creD

adjacent to P24811, the p8A plasmid was oligolabelled and used to probe the chromosome ll

specific cosmid library, The only positive hybridisations were to the cosmids already known to

contain creC and p8A, P24E 1 1 and P23004 (Todd et a/, , 2000), This indicated the presence of a

gap in the cosmid library, which may contain creD, The partial complementation on acriflavine

medium of L21808 and L2-Sal transformants is therefore the result of a different region of the

genome than that seen in pBd transformants, as these regions do not overlap. Hence it would

appear that there are a number of regions in the genome that can partially suppress the

acriflavine resistance phenotype due to creD34 mutation,

Since the walking approach in the chromosome ll cosmid library was shown to be

impossible due to the presence of gaps, the pBc region was radiolabelled by PCR and used to

probe an A, nidulans BAC library (Zhu ef a/., 1997) to identify a large region likely to contain creC

and creD. Forty-eight BAC clones hybridised to the pBc probe, including BAC22E20,BAC22E20

was shown to complement the creD34 phenotype on complete medium containing acriflavine and

on glucose medium with pyrrolidinone as a nitrogen source (Figure 3,5). To identify cosmids that

were contained within the region spanned by the complementing BAC, BAC 22E20 was

oligolabelled and used to probe the chromosome ll specific cosmid library. Again, only the

cosmids known to contain pBc (and creO) hybridised, indicating that there are large gaps in the

cosmid library, As BAC 22820 complements the creD34 phenotype, and yet does not conespond

to any cosmids other than the creC containing cosmids shown not to complement, the

chromosome ll specific cosmid library does not contain creD, The cosmid library was therefore

not a useful tool in identifying creD, and the BAC 22820 became the new starting point for the

identifìcation of creD via complementation,

3.4 ldentification ol creD within BAC22E20

The BAC 22820 contains approximately 70 kb ol A. nidulans genomic DNA, ln order to

refine the complementing region BAC 22820 was digested with Nof/ and religated to excise an

approximate 40 kb Nof/ fragment, resulting in plasmid 22Notrel1 which contains 30.79 kb of

genomic DNA. Plasmid 22Notrel1 was used to hansform acreD34; riboB2 strain and shown to

complement [he creD34 mutant phenotype on medium containing acriflavine and also on D-

glucose medium with pynolidinone as a nitrogen source to wild{ype levels (Figure 3.5),

Preliminary restriction analysis of 22Notrel1 was underway when the sequence of the A. nidulans

genome became publicly available via the Whitehead lnstitute's Center for Genome Research

website ( ), This resource

was utilized to analyse the A, nidulans genomic sequence contained within 22Notrel1. The

41

Chapter 3: Clonino and molecular analysis of creD

A

B

c

wild-type t22820 t22Notrell IAPY creD34

Figure 3.5 Complementation of the creD34 mutant phenotype. All strains were

grown at 37"C on either (A) 1% complete medium for 2 days (B) 1% complete

medium plus 0.001% acriflavine for 2 days or (C) 1% glucose plus 10 mM

pyrrolidinone medium for 3 days. Each strain is identified in the key below the plates,

with transformants of the creD34 strain designated tX, where X represents the

transforming DNA. Each transformant shown is representative of a number of similar

transformants generated in the same transformation experiment.

42


commercially available sequencing primer SP6 was used to sequence in from one end of

22Notrel1 to locate the region within the A. nidulans genome (Figure 3.6) as the SP6 promoter is

found in the BACwich vector used to constructthe BAC library (Zhu eta1.,1997). The 30,790 bp

Hindlll - Notlgenomic DNA fragment of the 22Notrel1 plasmid was searched for open reading

frames longer than 20 aa using the NEBcutter ORF programme (see Section 2.3.12), and reading

frames longer than 100 aa were analysed against the NCBI database using BlastX, Known and

hypothetical genes identified were roughly mapped within the 30 kb insert (Figure 3.6). The

GONf-like and YSC94-like genes were contained within 22Notrel1 (see 3,2.3), which is not

unexpected as BAC 22820 was identified by hybridisation to sequence within the YSCS4like

gene (pBc), As the pB3 plasmid contains these genes and was shown not to complement the

creD?4 phenotype (Figure 3.3) only putative genes downstream of this region were of interest.

fhe CBHP2)ike and ZDSAike genes had been previously shown to be within p8A (R. Sladic,

unpubl. dafa), which was shown here not to complement the creD34 phenotype (see 3.2,3, Figure

3.3). The remaining five regions containing large open reading frames within 22Notrel1 were

chosen for PCR amplification to test for complementation of the creD34 mutant phenotype as

they were sufficiently far away lrom creC according to expected distances from genetic mapping

comparisons to physical maps.

3.4.1 Complementation analysis of five regions within 22Notrell

Within the 22Notrel1 plasmid the five regions containing large open reading frames not

previously tested for complementation were designated APY, PH, mid, ST and novel (Figure 3.6).

The APY region consists of four adjacent ORFs (51 aa, 186 aa, 143 aa and 292 aa in length) that

show similarity to the arrestin domain and PY motif containing proteins, Rodlp and Rog3p from

S. cerevisiae, and both the 51 aa and 186 aa ORFs contain an arrestin domain as determined by

a conserved domain search in NCBI (http://www.ncbi.nlm.nih.goviStructure/cdd/wrpsb,cqi).

Primers APYF and APYR were designed to flank this hypothetical gene with at least an additional

600 bp upstream and downstream of the proposed gene starl and stop codons, to produce a

3328 bp PCR product. The PH region consists of a single 402 aa 0RF that contains a pleckstrin

[omology (PH) domain as determined by a conserued domain search using NCBI, and is most

highly similar to the N. crassa hypothetical protein E4A28289,1. Primers PHF and PHR were

designed to flank this hypothetical gene with an additional 1 kb of sequence upstream of the

proposed start codon, and 660 bp downstream of the stop codon, and result in a 2896 bp PCR

product. The ST region consists of two ORFs (93 aa and 358 aa) that are both highly similar to

43


smftnpercrtingtransfonmntsNH ENE H{r

BpiCz:P0 119

N E H

22Nürel1 4lB1

EN E

PBA u60

<-_><-___>ffi{|Yffi4 æfp N

H

+ APT

-> Pt-l

<( Íid<- sl

< ncvel

2J14

016

u25

0121

a41tò

Figure 3.6 A schematic representation of the hypothetical genes within plasmid22Notrell. Relevant Nof/ (/\/), Hindlll(F/) and Eco1l(E) sites are indicated on the BAC

22E20 that were used in lhe construction of the plasmid 22Notrel1 and orientation ofthe pBA plasmid, The bold blocks indicate pBACwich vector sequence, with the SP6primer site indicated by the block arrow. Bold arrows indicate the five regions

designated APY, PH, mid, ST and novel, amplified by PCR (see materials andmethods, Table 2.3) that were tested for complementation of the creD34 mutantphenotype, with the numbers of complementing transformants for each PCR product

indicated. Slim arrows indicate the hypothetical genes, GONf-like (5. cerevisiaeYAL048C), YSC&4-like (S. cerevisiae YHR016C), CBHP2-like (S. pombe

SPBC14F5.12c) and ZDS2like (5. cerevisiae YML109W), found within the p8A region

and shown not to complement.

44

Chapter 3: Cloning and molecular analysis of creD

the putative membrane transporter C4C36925,1 from S, pombe, with the 358 aa ORF containing

a sugar transporter (ST) domain as identified by a conserved domain search using NCBI. Primers

STF and STR were designed to flank this hypothetical gene with an additional 1,5 kb upstream of

the start of the first ORF, and 600 bp downstream of the putative stop codon, and produce a 3504

bp PCR product, The mid region consists of three adjacent large ORFs (159 aa, 171 aa and 282

aa in length) that have no conserved protein domains or highly similar sequences in the NCBI

database, Primers midF and midR were designed to flank these three ORFs with an additional

1.5 kb upstream of the start of the first ORF, and 500 bp downstream of the end of the third ORF,

and result in a 3857 bp PCR product, The novel region consists of a single 300 aa ORF, which

has no highly similar sequence in the NCBI database as determined from BlastX analysis, and no

conserved protein motifs. Primers novelF and novelR were designed to flank this ORF plus an

additional 500 bp downstream of the stop codon, and result in a 1418 bp PCR product. Using the

A. nidulans genome database, ORFs upstream of this 300 aa novel ORF were analysed to

identify a possible homologue, but immediately upstream ORFs were also not similar to any

sequence in the NCBIdatabase.

These five regions, APY, PH, ST, mid and novel, were amplified by PCR and the

resulting products were used to co-transform a creD34; riboB2 strain with the nboB* containing

plasmid pPL3 (Figure 3.6), The APY PCR product complemented the creD34 mutant phenotype

on both complete medium containing acriflavine and on glucose medium containing pyrrolidinone

as a nitrogen source to wild-type levels, whilst none of the other regions resulted in

complementation (Figure 3.5), Therefore, the APY PCR region that encodes a protein with

similarity to the Rodlp and Rog3p proteins from S. cerevisiae complements the phenotype due to

the creD34 mutation.

3.5 Molecular analysis of creD

3.5.1 Sequence analysis of creD and identification of the creD34 mutation

Primers were designed using sequence data from the Whitehead A. nidulans genome

database to span the length of the 3328 bp complementing region encoding the arestin domain

and PY motif containing protein. Genomic DNA from this region was amplified via PCR using

these custom oligonucleotides, and the resulting PCR products sequenced in both directions.

CreD is encoded within a 2028 bp sequence which was found to contain four introns, as

determined by sequence analysis of PCR products amplified from cDNA, and the intron

sequences conformed to the consensus 5' and 3' junction (GT-AG) and lariat (RCTRAC)

45


TTTCTGCTCGTCTTGGTCTTTCTACAGTCTGTCTACAGTCTGTCTATTAGTCTATCTGTTAGTCTGTCGTTGTCTATCTATTATCTATCTATTGTCTGTCGATTGTCTGTCGATTTGTCTGCTTGCCCTGCTTGAGGCTTCCÄACGGGTGTGAATCAGGGTTGTCÀÀTCAGCCGCTTTCTTTCAGCGCCGCGCTTTGCGCTCTAGCGCATGTTCGTCGAGATCGGTTATCGATTCCGGTGCAGAACA.ATACAGAÀGTAÃ.AATTTACCAGCTTAGTCAAÀCAGCATCGCATCÀTTCACTCCATACCCGTCGCCACAGCCCGTGCCGATÀÀAÀÀGACCCATCGCTGAGTGACGGAÃACGGCTGTTTTGGACCCTCCCCACTTCåÀTTCATATCCTGGTGGCACCCGAÀGATCGGTGCTCAGTGCCCTATCTGTGTCATACTTTTTTTTTTCCCTTTTTTCGCTTTACTCCGCCAGCCTTGGTTCCTATATTTCAGCÎGGTC

CÄCCCCCCTTGTGAGTGCGATÀGCACCTCTCCCCTTCGCACCGTTTCTGTTGGTGCTCATACTTTCTTGTTGCTGCTCTGAGTCGCCATGGCGCTCAGCTTCTTCAGCGGAGGCGGCAGTGCAAGTCATGCÃ.AÀGTACTTTGATATCCGgtgagEt EctgccatctcggaacttsMALSFFSGGGSASHAKYFD I R

cggEcct Eggcccgaact ctgcEaaEccgtccc EccgctgtcÈagACTCGACGAGGACTACATTGTTTTTCGTGGAGGTGAGCAAGALDEDYlVFRGGEQE

-523-436-349-262-L75-ss-L

!74

26].

348

435

522

609

1

22

36

65

71

L02

131

AGCCGCCAGTGCCCATCTA.AGCGGAAAGCTCGTTCTCTGTGTGTCGGAGCCGATÀTCTÀTCAAÀCATATTCGGTTGCÀTCTCACTGGAAS

gtaagtgcagcat EcEtggcggggt EacttcggtgaatggtctgcEgacaatggaEÈtcactag

TCCCATCGAGCTCTGCCGGCC,GCGGGCGCAAGAACTGGAGGGAGCGCGTATTCTACGAGAÀGACCTGGAÃATTCAGAGACGCGGGCA

GGCTCTCGGATACCTACGTCACGTATCGTTTCÀAGGCCGA.AATÀGGCCGCÀAGTATGCGAAGGACATCGTTGTTCGACGGCCGCTCC

l1) =creDiaGCATCATCCGCACCCTGGAGTCTÀGCGCCCTGGAÀCTTTCACACGCTATGgET EgEgatt.ccatcctcggtaEgacggtcacaEgcg

AI,EI,SHAM

GAGTATCCGGGTCGACTTCÄÀGCTGATCCCTCTCCTTA.AAGGACTTGGCATTGGTCAGATTATCTCGCAGCTCATAGAGACGCACGA

CGAGGAAÄACTCCTTAGAÀÂTÀATAGACGAÄGCTGCGGAÀC'GGTTTCAGTTCTCGCGTACGCTCGACCTGCCCAA.AACCTTGACGCG

ATGTCTGCAGGATACAGAEÂCCÀGGGGGATCÀÃAGTGAGACACAAGTTGAÂATTCAGAGTGCÀACTGCTCÀACCCTGATGGGCATAT

tacggaaaggaÈagcctcctgct gtsatggatgcgc Eaaccacagtacag

ATCTCCCCGAÀCCTCGCGATTGATGACAÀCAÀCAÀCCTTGTCGATTCAAGCCCTCAGACGACCCAAAGAGCGCTTGATGATCTCGCTI D D N N N I, V D S S P Q T T O R A T, D D IJ A

CAGCAAGCGCCTCCTTTGTATGGGGAÀCACCAGTTCGATCAGCTGTACAGCGÀÀGTTGATCCTTCCGGTTACCGCACCCCTGGTCCT

GGAAGTGGTCCTGGTACACCTTTTGGCACGCTTAGTCGCAACCTATCCGCCGAGAÀCCTTGCATCCATGAATGCTATCACCCACACCG S G P G T P F G T I, S R N I, S A E N I, A S M N A I T H TGACATCTCCGCTTCAGCCTTGCÀCCATCGCTTGGTAAÀCCTTGATTTGCGCGGACATGGTCGGGTATCGGCATCAGAÀCATGATCATD I S À S A I, H H R ], V N TJ D L R G H G R V S À S E H D ¡T

CTCGGTGTTCCTTCAGACÃACGGGCCCCCCTCGGGCTCTAACACACACGGCTCCAACACCCACGCACCCGGAAGCCCAGAÄCTTTCG

CTCÀGTCGCGTGCCAÄGCTATTCCACCGCTGTCCGTTCATCTGTTCGTCCACATGACAGCGATCTGCCAGATTATCAC'GCCGTTGTT

L s n v li]s]]l s r A v R s s v R p H D s D L þ:J:g e A v vGCTGAÄACTGÎCCÀTATGTCAGCCCCCCAATCCCCCCAGCAAGCGCACATCCGGGGGTCTGGTACTGGTCGCGGATCGGACTCTTAT

TTTAGCGCACCCÀTGGATTTCTTTCATCGACCTGCTTTTCTCCATTCCCGÄÀGTCATAGTCATTCCGACGACGAACGCAGGATTCGT

TCCTGAÄACAGATACCCGCCGCCCTATTCÀACGGCCCGTTÀTTGAGCGATTCTAACCACCGCTGTTCCATTGAAGCCGGGATTCCACCACATATÄÄTATTCCTTCGAÀCGACCACGTTACCAGCGCGGGACGCTGATTTGCATATATTTATAGAATCTCCTAÀTCTTCCACTCAACåÄÀGTACCCÀGAGGATCTCGTTTTTGTATCTATATTCCAACATGTTCCTCTGTAÀÀCGCGATTGTTTTGAGATCÀGGGAAGGTCTACTGGCGTTTTAÀCATAGACGGACTGGAGTGGAAÀTTATGCCAATAAÄÀAGGGATGATGGAAÀTCAATAÀTGACTCCTTGCTATGAATTÄAGTATCÀTATATGAÀCTATGGATATGÄAGGGATÀÀGCTATAAGATTGAGACTGCTGCAAGGTCTCGGCAGTAGACGATCCCCCTTGTTGACAGAGCCATCTAGACATTCTTGCGTGGTTGTCGATCGACÀTATTCAGCGGÄTCTGAAGATCTTGCCGCAGAÀTTCCATTTGCGATCTGGCÀÄAGCCACATCTACTGGTGCGAÀACTGATTATCGGGGTGTAAÃ.ACTAGCCåÀCCTCGATCTAAGCCTGTACCTGAÄGATGGTTCCCGCAGAÃÃGAGCGTCGAT CCGACA.ACACAGCCÀCACCT

160

yt6

20r

230

259

288

31?

329

358

3A't

416

445

474

503

532

561

590

696

7S3

870

957

LO44

1131

1218

1305

L392

r479

1566

1653

r7 40

LA27

L9!4

2001

2088

SVENI

GEHQRDQLYSEVDP SGYRTPGPooA

I, GV P S DN G P P S G S N T HG S N T H A P G S P E I, S

RRA SD EDVHDN I P S GMAT P F I P H gAE I, E T

AETVHMSAPQSPOQAHI RG SGTGRGSDSY

F S A PMD F FHR PÀF I,H S R SH S H S DD E R R I R

I.TQARGRA*2r752262234924362523261026972742

Figure 3.7 Location of the creDs4 mutat¡on within the creD gene. The complete sequence of

the creD genomic region is shown. The four introns are depicted in lowercase letters and lariat sequences

are underlined, A putative TATA box is underlined and in boldface type, and CAAT motif bolded and

italicised. The amino acid sequence is shown in single letter code beneath the DNA sequence, with

numbered amino acid residues indicated on the left-hand side, and numbered nucleotide positions

indicated on the right-hand side, with the first nucleotide of the start codon as +1. The position and nature

, The PP)fl motif is highlighted yellow and boxed in

a bold line, and the minimalcore P)ff motifs are highlighted yellow and boxed in a broken line. The creD

sequence has been submitted to the GenBank database under Accession number 4Y458430.

46

Chapter 3: Cloning and molecular analvsis of creD

sequences for filamentous fungal introns (Gurr ef a/., 1987) (Figure 3,7). The predicted translation

starl point of CreD shows similarity to the Kozak consensus sequence (Kozak, 1987), and a

putative TATA box is located 103 bp upstream of the ATG, and a CAAT motif 206 bp upstream of

the ATG. The creD gene encodes a 597 aa protein that contains an arrestin_N domain (aa 39 -

168) (PF00339), an arrestin_C domain (aa 181 - 334) (PF027252) (SMART protein motif

analysis programme: Schultz et al., 1998), a single PP)ff motif (aa 361 - 364) and two P.,\A

motifs (aa 507 - 509 and 525 - 527).

Particularly in view of the partial suppressors initially identified, lhe creD gene was

sequenced from PCR products produced from genomic DNA obtained from a wild-type strain and

a strain with the creD34 mutation to identify the molecular nature of the creD34 mutation and

confirm that this gene was indeed creD. Primers were designed using sequence data from the

Whitehead A. nidulans genome database (see Table 2.3). The creù and creD34 alleles were

sequenced in their entirety and creD?4 was found to contain a Gto-T transition at nt 642 that

causes the codon for glutamic acid at aa170 to become an ochre stop codon, thereby truncating

the remaining 427 amino acids. This mutant gene product lacks the PY motifs and the arrestin_C

domain (Figures 3.7 and 3.8).

3.5.2 Bioinformatic analysis oÍ creD and identification ol apyA

Analysis using BlastX revealed CreD was most similar to the hypothetical protein

SPAC31A2.12 from Schizosaccharomyces pombe (1.8e-60), with the most similar analysed

proteins to CreD being Rodlp and Rog3p from S. cerevisiae, which both contain an arrestin_N

and arrestin_C domain, two PP)ff motifs and a P)ff motif. BestFit alignments of CreD and

Rod1p, and CreD and Rog3p, indicate they share 32.8% aa identity and 55.0% aa similarity, and

343% aa identity and 56.9% aa similarity respectively, across the length of the proteins (Figure

3.94). As there were two such highly conserved sequences to CreD in S. cerevisiae, creD was

used to search the A. nidulans genome database and a second gene, apyA, encoding a

hypothetical protein containing an arrestin_N and arrestin_C domain and flP,Ìf, motif was

identified on chromosome Vl (locus 4N3265.2) (Figure 3.8), apyAwas amplified from cDNA using

PCR and sequenced, and the sequence concuned with the A. nidulans Whitehead genome

database prediction of an 1107 bp ORF interrupted by four introns. The apyA cDNA sequence

data was entered into the GenBank database under the Accession number 4Y460113. The

hypothetical ApyA is 368 aa in length, and shares 44.2% aa identity, and 64.9% aa similarity with

CreD, BestFit alignments of ApyA and Rod1p, and ApyA and Rog3p, indicate they share 29$%

aa identity and 54.0% aa similarity, and 31 .3%aa identity and 55.5% aa similarity respectively,

47


DF1t creD34 DR1ts

CfeD 1 se7

arrestin N

domain

arrestin C PP)trdomain motif

P)ffmotifs

ApyA upvtl aovARË'1 368

arrestin N

domain

arrestin Cdomain

PP)ffmotif

Figure 3.8 Schematic representation of CreD and ApyA. The diagonally striped

box indicates the anestin_N domain, the gridded box represents the anestin_Cdomain, the filled box represents the PP)ff motif and the open box represents the

P)ff motifs. Block anows indicate the primers used to amplify regions of CreD andApyA ligated into the pTRG vector (see materials and methods). The CreD34 protein

is truncated at the position marked with an asterisk.

48


across the length of the proteins (Figure 3.94). To determine the phylogenetic and evolutionary

relationship between these genes, maximum parsimony analysis was undertaken which included

arrestin domain containing proteins lrom Aspergillus fumigatis (Apy1), Neurospora crassa (Apy1)

and S. pombe (Apy1 and Apy2), identified by Blast searches in the respective genome

databases. lnterestingly, the two S. cerevisiae proteins grouped strongly together (with 96%

support of bootstrap replicates), indicating BOD| and ROG3 are likely to be paralogous genes

resulting from a gene duplication event within the S. cerevisiae lineage (Figure 3.98). Similarly,

creD and apyA appear to have arisen due to a gene duplication within the A. nidulans lineage

(Figure 3,98), a duplication event that did not occur in N. crassa as only a single protein

containing an arrestin domain was identified in the complete genome sequence of N. crassa.

These results account for the almost identical similarities of ApyA and CreD to Rodl p and Rog3p.

3.5.3 Multiple copies oÍ apyA do not compensate for the absence ol creD

Since the creD74 mutant phenotype is apparent in an apyA* background, the two

proteins cannot simply have identical functions unless their concentration is limiting. To

investigate whether multiple copies of lhe apyA gene can compensate for the lack of a functional

creD gene, a genomic clone of lhe apyA gene was generated by cloning the PCR product

generated using primers apyACF and apyACR (see Table 2.3), and DNA of fosmid 8052C12

(known to contain apyA) as the template, into pBluescript. This clone, pBapyA, was introduced

into a creD34; riboB2 strain by co{ransformation with pPL3 containing the riboBr gene. The

transformants were analysed by hybridisation of Southern transfers, and strains with a low

number (2) and a high number (5+) of copies of apyAwere identified (Figure 3.10). These strains

were tested for sensitivity to the presence of acriflavine in complete medium and growth on

pyrrolidinone as a nitrogen source, and they showed the same phenotype as creD14 (Figure

3.1 1). Thus, multiple copies of apyA cannot compensate for the absence of functional creD,

3.6 Proteins that interact with CreD and ApyA

3.6.1 Testing protein-protein interactions between CreD and CreA, ApyA and CreA

The Stratagene BacterioMatch lbacterial-2-hybrid system (B2H l) is an easy and rapid alternative

to yeast two-hybrid systems for the detection of protein-protein interactions rn vivo. All screening

and validation steps are pedormed in bacteria. A protein of interest (the bait) is fused to the full-

length bacteriophage l,cl repressor protein (ì.cl) within the bait vector (pBT), and binds to the À

operator. The target protein is fused to the N-terminal domain of the cx-subunit of RNA

49


A

CreD

44.264.9

ApvA

34.956.9

29.954.0

32.855.0

31,355.5

Rodl p

44.0ô0.9

Rog3p

B

99 AfApyl

AnCreD

AnApyA

NcrApyl

ScRodlp

ScRog3p

SpomApyl

SpomApy2

91

68

42

96

Figure 3,9 Evolutionary relationship between CreD and other fungal arrestindomain containing proteins. (A) Amino acid identities and similarities (percent)

between the anestin domain containing proteins from A. nidulans, CreD and ApyA, andS. cerevisiae, Rodlp and Rog3p, as determ¡ned from pair-wise BestFit alignments.Amino acid identities are in bold, above amino acid similarities in normal type,

corresponding to the two proteins indicated by the nearest double-headed arrow. (B)

Maximum parsimony analysis of fungal arrestin domain containing proteins from A.

nidulans, AnCreD and AnApyA (AN3265.1), A. fumigatr's, AfApyl (AFGDP80TR.69), N.

crassa, NcrApyl (EA428290.1), S. cerevisrae, ScRodlp (YOR018W) and ScRog3p(YFR022W), and S. pombe, SpomApyl (SPCC584.15C) and SpomApy2(SPAC31A2.12). Bootstrap values are adjacent to each internal node, representing thepercentages of 1000 bootstrap replicates.

50


WT 034 A1 A2 A3 A4 A5 A6

0 0 344-57-9 I

Figure 3.10 Determining apyA copy number in tapyA transformant strains via

Southern analysis, Autoradiograph exposed overnight to a Southern filter probed with

apyA that contained genomic DNA from WT, a wild-type strain, D34, the creD34 parent

strain, and A1-A6, six individual cotransformants of riboBr and apyA. The anow indicates

the endogenous copy oI apyA. Additional copy numbers of apyA were estimated by the

number of additional bands and strength of hybridisation. Phosphorimaging quantitative

analysis of the additional bands relative to the level of hybridisation to the endogenous

apyA band in each individual transformant track was also performed to help determine

additional copy numbers of apyA in each transformant, and these estimates are shown

below the autoradiograph.

51


rt

dF

td},

#

dF

#Complete medium

B

Complete medium + acriflavine

tapyAl lapy\2 tapyA3

#' +-' &. tapyA4 tapyA5 tapyA6

,& wild{ype creD}4

G D

Glucose + pyrrolidinone

Figure 3.11 Multiple copies ol apyA does not compensate for lack of creD. All strains

were grown at 37"C on either (A) 1% complete medium for 2 days (B) 1% complete

medium plus 0.001% acriflavine for 2 days or (C) 1% glucose plus 10 mM pyrrolidinone

medium for 3 days, The strains are indicated by the key (D), Six individual riboB* apyA

cotransformants are designated tapyAl -6,

52


polymerase within the target vector (pTRG), When the bait and target interact, they recruit and

stabilize the binding of RNA polymerase close to the promoter and activate the transcription of

lhe ampr repofter gene, A second reporler gene,lacZ, is expressed from the same promoter and

provides an additional screen to validate the bait and target interaction

To investigate whether CreD or ApyA interact with CreA, the bacterial-2-hybrid system

(B2H l) was used to detect protein-protein interactions. The entire coding region of apyA cDNA

was ligated into the target vector, to produce the construct pTRGapyA, and the majority of the

coding region ol creD cDNA, incorporating amino acids 31 to 580 that encompass the arrestin

domains and PY motifs, was ligated into the target vector to produce the pTRGcreD construct,

The previously made pBTcreA construct incorporates aa 139 to 416 of CreA into the pBT vector,

which includes all of the CreA protein beyond the DNA binding region (R. Lockington, unpubl.

data). All vectors were tested for auto-activation of the reporter system, and deemed suitable for

use in the B2H lsystem as no auto-activation was observed. The pBTLGF2 and pTBGGALIIp

constructs provided with the B2H I kit served as a positive control. Neither CreD and CreA, nor

ApyA and CreA directly interacted in this system.

3.6.2 ldentification ol hulAand testing HulA for interactions with CreD and ApyA.

The Rodlp and Rog3p proteins have been shown to interact via their PY motifs with the

WW domains of the ubiquitin ligase Rsp5p in S. cerevisiae (Andoh et al., 2002). ln order to

identify the,4. nidulans homologue of the Rsp5p protein, the,4. nidulans genome was searched

using the Rsp5p sequence in a tBlastN search, and six HECT gbiquitin ligases were identified

and designated as follows; hulA (4N1339.2), hulB (AN0444.2), hul0 (4N1746.2), hulD

(4N1874.2), hulE (AN1966.2), and hulF (4N3999.2). Rsp5p was used to also search the S.

cerevisiae genome via a tBlastN search, and five HECT ubiquitin ligases were identified as

follows; Rsp5p, Tom1p, Hul4p, Hul5p and Ufd4p. Phylogenetic analysis was undeftaken on all 11

proteins to identify orthologous genes from the two species, and the Rsp5p homologue in A.

nidulans was identified as the hypothetical protein HulA (4N1339,2), strongly suppofied with

1 00% of bootstrap replicates of the Neighbour-Joining dendrogram (Figure 3.12),

Like Rsp5p, the A. nidulans HulA protein contains a HECT ubiquitin ligase domain,aC2

domain and three WW domains. To examine whether the interactions between the PY motif

containing proteins and the WW domains of HulA also occurred in A. nidulans, constructs were

made to test the direct protein-protein interactions between CreD and HulA, and ApyA and HulA

via the BacterioMatch I bacterial-2-hybrid system. The pBThulA plasmid was constructed by the

PCR amplification of the A. nidulans hulA gene from cDNA using the primers BThulAFRl and

53

Chaoter 3: Clonino and cular analvsis ol creD

100 AnHulA

100 ScRspSp

AnHulB89 100 ScTomlp

AnHulE

BO AnHulC

98 ScHu14p

AnHulD

100 ScHul5p

AnHulF

100 ScUfd4p

0.2

Figure 3.12 Evolutionary relationships between the HECT ubiquitin ligases inS. cerevisiae and A, nidulans. Phylogenetic analysis using the neighbour-joiningmethod of all HECT ubiquitin ligases identified in the A. nidulans and S. cerevisiaegenomes. The prefix An denotes an A. nidulans protein, and the prefix Sc denotesa S. cerevisiae protein, Bootstrap values are adjacent to each internal node,

representing the percentages of 1000 bootstrap replicates. The scale bar

represents 20 changes per 100 positions,

54


BThulARBam, incorporating amino acids 218 to 445 that encompass the three WW domains of

HulA into the pBT vector. The ApyA protein was shown to interact with the WW domains of HulA,

as indicated by growth on carbenicillin containing medium and validated by a blue colour reaction

on X-gal indication plates (data not shown). However, the CreD protein did not interact with HulA

in the B2H lsystem.

The BacterioMatch ll bacterial-2-hybrid system was released in late 2003, and is

compatible with plasmids from the B2H I system, but utilizes a different reporter system that has a

reduced background and is thus more sensitive for detecting protein-protein interactions. The

new H/S3-aadA reporter cassette allows the detection of protein-protein interactions by

transcriptional activation of the H/S3 reporter gene, which allows growth on minimal medium

lacking histidine. The competitive inhibitor of the His3 enzyme, 3-amino-1,2,4-triazaole (3-AT),

was used to reduce the background, Potential positive interactions can be verified by the

secondary reporter aadA gene, which confers streptomycin resistance, To differentiate between

the positive and negative controls for the B2H I system, the carbenicillin concentration used to

test for protein-protein interactions was three times more than that recommended by Stratagene.

To confirm the positive interaction seen between ApyA and HulA, and to test the possibility that a

weaker interaction between CreD and HulA that may have fallen beneath background detection

levels in the B2H I system exists, the new B2H ll reporter cells were transformed to test for

interactions between these proteins. The ApyA protein was shown to interact strongly with the

WW domains of HulA, as indicated by strong growth on medium lacking histidine (H/S3

activation) and validated by resistance to streptomycin (aadA activation) (Figure 3,13), lnteraction

of the CreD protein with HulA was clearly detected, but it was weaker as indicated by weaker

growth on medium lacking histidine (+/- streptomycin) (Figure 3,13).

3.7 Discussion

ln the initial stages of this work, complementation of the creD34 mutant phenotype was

only assessed on complete medium containing acriflavine, On this test, pBA as well as other

regions of the genome not in the region of creD (eg, cosmid 121808) apparently complemented,

For p8A, this region was narrowed down to a 701 bp region that contained the regulatory regions

of the divergently transcribed GONl-like and YSCS4like genes, but sequence analysis showed

no sequence differences between the wild{ype and creD34 strains. Thus certain regions of the

genome, when in high copy number, can suppress the acriflavine resistance phenotype conferred

by the creD34 mutation. However, when these transformants were tested on glucose medium

containing pyrrolidinone as a sole nitrogen source, no complementation was observed, and they

55

Chapter 3: Cloning and molecular analysis of creD

A

Non-selective

medium

B

Selective Screening

(3AT)medium

c

Dual Selective

Screening (3AT +

streptamycin) medium

D

*ve D1 D2 D3 D4 D5 K-ve D-ve

H-ve

+ ve A1 Æ A3 A4 A5 K-ve A-ve

Figure 3.13 lnvestlgation of protein-protein interactions between CreD and HulA, and ApyA

and HulA as determined by the BacterioMatch ll bacterial-2- hybrid system. (A) Non-

selective screening (minimal) medium supplemented with 25 pg/ml chloramphenicol and 12.5

pg/ml tetracycline. (B) Selective Screening Medium containing minimal medium plus 5 mM 3-

amino-1,2,4-triazole (3-AT), 25 pg/ml chloramphenicol and 12.5 pg/ml tetracycline (C) Dual

Selective Screening Medium containing minimal medium plus 5 mM 3-AT and 12.5 pg/ml

streptomycin, 25 ¡rg/ml chloramphenicol and 12.5 pg/ml tetracycline, (D) Key to strains: +ve

indicates the positive controlstrain, cotransformed with pBTGLF2 and pTRGGallp. D1-5 are five

independent strains cotransformed with pBThulA and pTRGcreD. K-ve indicates a negative

control strain, cotransformed with pBT and pTRGGall lp. D-ve indicates a negative control strain

cotransformed with pBT (no insert) and pTRGcreD. A1-5 are five independent strains

cotransformed with pBThulA and pTRGapyA. A-ve indicates a negative control strain

cotransformed with pBT (no insert) and pTRGapyA, H-ve indicates a negative control skain

cotransformed with pBThulA and pTRG (no insert), All plates incubated at 37oC overnight.

56


grew as poorly as the creD?4 mutant strain (Figure 3.3). These early experiments were utilising

the ordered chromosome ll specific cosmid library ol A. nidulans, however it was shown here that

this was not suitable for the identification ol creD as the cosmid library is unordered in this region

and contains large gaps.

Fhe creD gene encodes a protein that contains an arrestin_N and arrestin_C domain, a

PP)ff motif and two P)ff motifs, and is highly similar to the Rodlp and Rog3p proteins from S.

cerevisiae. The presence of two such similar sequences lo creD in the S. cerevisiae genome led

to the search for another arrestin domain containing protein in A. nidulans, which identified a

gene we have designaled apyA, encoding an arrestin_N and arrestin_C domain and PP,XY motif

containing protein. ln S, cerevlslae Rodlp and Rog3p have been shown to interact with the

ubiquitin ligase Rsp5p, and so to determine whether this interaction occurred in A. nidulanslhe

homologue of Rsp5p was identified, and the gene encoding this HECT gbiquitin ligase was

designated hulA. CreD and ApyA were tested for interactions with HulA via the bacterial-2-hybrid

system, and strong interaction was found between ApyA and HulA, with weaker but clear

interaction between CreD and HulA.

Rodlp and Rog3p from S. cerevisiae are the two most highly similar characterized

proteins to CreD and ApyA, and both of these proteins contain an arrestin_N and arrestin-C

domains, two PP)ff motifs and a P)ff motif. PY motifs [PP)Cí] are small proline rich sequences

that are commonly bound by WW domains (Chen and Sudol, 1995), and P)ff has been shown to

be a minimal basic core that can bind WW domains with reduced affinity (Chen et al., 1997).

ROD| was identified in a high copy plasmid library screen selecting for increased resistance to o

- dinitrobenzene (oDNB). The Rodlp protein also plays a physiological role in the resistance to

calcium and zinc (Wu ef a/., 1996). Loss of Rodlp causes cells to become hypersensitive to o

DNB, calcium, zinc and diamide (Wu ef a/., 1996), HOGS was identif ied as a suppressor mutation

of the temperature sensitivity found in a mckl mdsl double mutant strain, where mckl and mdsl

encode homologues for glycogen synthase kinase 3 (Andoh et a1.,2002). Rodlp and Rog3p

share 44% sequence identity with each other, and both of these proteins have been shown to

interact via their PY motifs with the WW domains of the E3 ubiquitin ligase Rsp5p (Andoh ef a/.,

2002\. The Rsp5p ubiquitin-protein ligase contains a HECT ([omologous to the !,6-Associated

Protein Carboxyl Terminus) domain that catalyses ubiquitin transfer, and three WW domains.

WW domains are protein modules approximately 40 amino acids in length which contain two

highly conserved tryptophans about 20 - 23 aa apart (Bork and Sudol, 1994). These small

domains fold in a three stranded, anti-parallel B-sheet, which creates a hydrophobic pocket for

binding proline rich ligands motifs. WW domains of the Rsp5p type bind PP)ff motifs (Chen and

57

Chapter 3: Cloning and molecular analvsis of creD

Sudol, 1995). Rsp5p is not only involved with protein degradation, but other cellular functions

such as transcriptional regulation and transcriptional-coupled repair (Chang et al.,2000), and is a

trans-activator for the steroid-hormone receptor family of transcription factors (lmhof and

McDonnell, 1996). The human homologue of Rsp5p, Nedd4, has been shown to regulate the

turnover of the epithelial plasma membrane sodium channel (ENaC), Mutations of the PY motif in

the ENaC disrupt the interaction with the WW domains of Nedd4 and results in Liddle's

syndrome, a severe hereditary form of hypertension in humans (Schild ef a/., 1996).

The CreD protein is predicted to be involved in the ubiquitination aspect of the regulatory

network involving CreA, CreB, CreC and AcrB from the analysis of the epistatic interactions of the

various mutation combinations (Kellyand Hynes, 1977; Boase eta\.,2003). The proposed model

of carbon catabolite repression regulated via CreA modification or stability involves the CreB

deubiquitination enzyme acting in a protein complex with (at least) the CreC protein to modify or

stabilize CreA under carbon repressing conditions (Lockington and Kelly, 2002). Strauss ef a/.

(1999) have also implicated protein modification or stability of CreA in the mechanism of carbon

catabolite repression. The CreBiCreC complex is predicted to have substrates additional to CreA,

as cre9and creC mutations have a broader range of mutant phenotypes under both repressing

and derepressing conditions than creA (Lockington and Kelly,2001). Both acrB and creD

mutations have been shown to suppress aspects of the creB and creC mutant phenotypes (Kelly

and Hynes, 1977; Boase et a1.,2003) implicating creD and acrB as having a role in ubiquitination.

The interaction in S. cerevisiae between Rodlp and Rog3p with the E3 ubiquitin-protein

ligase Rsp5p, and the presence of two homologous proteins, CreD and ApyA, in A. nidulans

would suggest that there might also be an interaction between CreD and HulA, and ApyA and

HulA in A. nidulans. Bacterial-2-hybrid (B2H ll)analysis of CreD and ApyA with the WW domains

of HulA from A. nidulans indicated that ApyA and the WW domains of HulA interact strongly, and

that CreD and HulA interact, but less strongly, in this system. The ApyA protein of A. nidulans

shares 44%amino acid identitywith CreD, and both contain an arrestin N and arrestin C domain

and a single PP)ff motif. CreD contains the low-affinity minimal core motifs, PSY and PDY, plus

the PP)ff motif, PPLY, whereas ApyA contains a single PY motif, PPAY, These different motifs

may have different affinities for any given binding partners, or interact with different parlners. The

introduction of extra copies otlhe apyA gene did not suppress the phenotype due lolhe creD?4

mutation, and thus in the absence of CreD the elevated levels of apyA expression cannot

compensate. Thus the ApyA protein of A. nidulans is a newly identified protein proposed to be

involved in the ubiquitination aspect of this regulatory network.

58

Chaoter 3: Cloninq and molecular analvsis ol creD

There are a number of HECT ubiquitin ligases in the A. nidulans genome, and since

CreD did not interact as strongly as ApyA with HulA, it is possible that CreD also interacts with

another HECT ubiquitin ligase. A BlastX search of the A. nidulans genome revealed the presence

of six such HECT ubiquitin ligases, and a BlastX search of the S. cerevisiae genome identified

five HECT ubiquitin ligases. Phylogenetic analyses on all 11 proteins revealed that each of the

five S. cerevisiae HECT ubiquitin ligases had a clear orthologue in A. nidulans. The additional

HECT ubiquitin ligase in A. nidulans HulE (4N1874.1) nay be a partner for CreD, creD and apyA

appear to be the result of a gene duplication event, so it is reasonable to think that they may have

evolved different specificities for the same substrates, as well as different substrates.

Both CreD and ApyA contain an anestin_N and arrestin_C domain. Arrestins specifically

bind to phosphorylated activated forms of their associated G protein-coupled receptors preventing

further G protein activation, often redirecting signalling to other pathways (reviewed in

Palczewski, 1994). This suggests that CreD is involved in signalling by recognising appropriately

phosphorylated substrates via its arrestin domains, The arrestin domain could target CreD and

ApyA to phosphorylated substrates, and then recruit a HECT type ubiquitin ligase to ubiquitinate

the substrate, providing a link between ubiquitination and phosphorylation in protein regulation

and stability. There is currently no mutant available tor apyA. The similarity in sequence to creD

would suggest a role in the carbon catabolite repression pathway, and the interaction with HulA

implies ApyA could also be involved in the ubiquitination aspect of the network that involves the

CreB/CreC complex.

59

Chapter 3: Cloning and molecular analvsis ol creD

60

Chaoter 4: Molecular characterization and analvsis of acrB

CHAPTER 4: MOLECULAR CHARACTERIZATION AND ANALYSISOF acrB

4.1 lntroduction

The acrB2 mutation was isolated in the filamentous fungus A. nidulans as a spontaneous

resistant sector on complete medium containing acriflavine in a genetic screen with the joint aims

of understanding acriflavine toxicity and obtaining extra tools for genetic mapping (Roper and

Kafer, 1957). As outlined in the previous chapter, lhe creD34 mutation also confers resistance to

acriflavine and was identified as a suppressor of the effects of the creC27 mutation. This aspect

of phenotypic similarity between acrB2 and creD34led us to analyze whether lhe acrB2 mutation

is also a suppressor of the phenotypes due to creB and creO mutations, which would indicate a

role for AcrB in a regulatory ubiquitination pathway that is required for correct regulation of carbon

metabolism.

4.2 Phenotypic analysis ol acrB2, acrBl4 and acrBlí

As selected, the three acrB mutant strains analysed, acrB2, acrÙ14 and acrBlí, were

resistant to the presence of acriflavine in complete medium, with similar levels of resistance

between acrB mutant strains and the creD34 strain (Figure 4.18). All three alleles conferred a

slightly altered colony morphology on complete medium, All three acrB mutant strains were more

sensitive to the presence of 11 mM molybdate in synthetic complete medium than the wild-type

strain, but the alleles were heterogeneous, with the acrB2 strain being the most sensitive to the

presence of molybdate, and the acrBl5 strain showing lesser sensitivity (Figure 4.1D). Thus the

effects of the acrB mutant alleles when the toxic compounds acriflavine and molybdate are added

to the medium are the same as those for creD34, and the opposite to the effects of creB and creC

mutant alleles which confer increased sensitivity to acriflavine and resistance to molybdate added

to complete medium (Arst, 1981; Hynes and Kelly, 1977;Kelly and Hynes, 1977).

When tested for their ability to utilize various carbon sources, acrB mutant strains were

found to have pleiotropic phenotypes with respect to carbon source utilization. They show

decreased ability to utilize a number of different sugars as sole carbon sources in comparison to

both the wild-type and the creD34 mutant strain, including fructose, cellobiose, trehalose and

starch. ln general, the acrBl4 strain showed a slightly more extreme phenotype than the acrB2

and acrBl5 shains on alternative carbon sources (Figure 4.|E-F; Table 4.1). The reduced growth

ô1

Chapter 4: Molecular characterization and analvsis of acrB

wild-type croD34 acrÙ2 acrB14 acrBlí

A, Complete medium

* *, *t t, B. Complete medium + acriflavine

C, Glucose + ammonium

4r D. Glucose + ammonium + molybdate

{F** E, Fructose + ammonium

F, Trehalose + ammonium

## G. Ethanol+ ammonium

** ffi,-- * H, Quinate + ammonium

*f l. Glucose + ammonium + allyl alcohol

J. Glucose + fluoroacetamide

lç I * K, Glucose + pyrrolidinone

Figure 4.1 Phenotypic heterogeneity among acrB mutant alleles. Strains containing acrB mutant alleles,acrB2 (strain acrB2G), acrB14 and acrB15, were grown at 37oC for 2-3 days, along with a strain containingcreD34 and a wild{ype strain on the following media: (A) 1% Complete medium; (B) Complete medium plus

0.001% acriflavine; (C) 1% D-glucose plus 10 mM ammonium L{artrate; (D) 1% D{ructose plus 10 mMammonium L{artrate; (E) 1% D-glucose plus 10 mM ammonium L{artrate plus 11 mM molybdate; (F) 1% D-

trehalose plus 10 mM ammonium L{artrate; (G) 0,5% ethanol plus 1OmM ammonium L-tartrate; (H) 50 mMquinate plus 10 mM ammonium L{artrate;(l) 1% D-glucose plus 10 mM ammonium L-tartrate plus 1OmM allylalcohol; (J) 1% D-glucose plus 10 mg/ml flubroacetamide; (K) 1% D-glucose plus 10 mM 2-pyrrolidinone.

62

Chapter 4: Molecular rization and analvsis oÍ acrB

Table 4.1 Phenotypic analysis of acrB mutant strains.

Growth condition Wild-tvpe creD34 acrB2G acrBl4 acrBl5

Complete 10 10 9 o IGlucose 10 10 10 10 '10

Cellobiose 10 10 5 5 5

Fructose 10 10 4 3 4

Galactose I B 4 3 4

Lactose I I 4 4 4

l\4altose 10 10 6 5 6

Raffinose 10 10 6 6 6

Starch 10 10 B 8 8

Sucrose 10 10 7 7 7

Trehalose 10 10 6 5 6

Xylose 10 10 7 7 7

Acetate 9 10 6 5 IEthanol 10 10 5 4 5

Slucuronate I I 7 7 7

Glycerol 10 10 I I ISuccinate 3 3 3 3 2

Quinate 10 10 I I IAcriflavine I I I 10 10

[4olvbdate 10 5 2 3 4

Fluoroacetamide 3 5 0 0 0

2.5 mM allvl alcohol 10 10 10 10 10

10 mM allvl alcohol I 9 10 10 10

ß-alanine 2 1 2 1 2

GABA I I 2 2 2

Pvrrolidinone I 8 2 3 1

Glutamate 8 8 4 4 4

Proline 8 8 4 4 5

ïhreonine 2 2 2 2 2

ß-alanine + qlucose 10 5 6 6 6

GABA + glucose 10 I 5 5 5

Pvrrolidinone + qlucose 10 1 3 5 7

Glutamate + qlucose 10 10 I o 9

Proline + qlucose 10 10 10 10 10

Threonine + qlucose 10 4 10 7 10

Glucose + acetamide 10 I 10 9 10

{cetamide 5 5 4 4 4

Acetamide + [NH4lzT 5 5 4 4 4

Slucose + acrvlamide 1 1 1 1 1

Table 4.1 Legend, For full genotypes, see Materials and Methods, lable 2.2. Plates were incubated at 37oC for two

or three days. Carbon sources were added to carbon free medium containing 10 mM ammonium L{artrate a|1.2%(acetate), 1% (D-cellobiose, DJructose, D-galactose, D-glucose, glycerol, lactose, D-maltose, D-raffinose, starch,

succinate, sucrose, D{rehalose, and D-xylose), 0.5% (D-glucuronate and ethanol) or 50 mM ((-)-quinate). Molybdate

was added to synthetic complete medium at 11 mM. Fluoroacetamide was added to 1% D-glucose medium at 10

mg/ml. Allylalcoholwas added to synthetic complete medium at 2.5 mM or 10 mM. Acetamide, B-alanine, GABA (yamino-n-butyric acid), L-glutamate, L-proline, 2-pyrrolidinone, and L{hreonine were added at 10 mM to 1% glucose

medium, and at 50 mM when added to medium without another carbon source. Ammonium L-tarlrate and acrylamide

were added at 10 mM. Growth is scored on a numerical scale with 10 the strongest growth, and 1 the weakest.

Scores are not directly comparable between plates.

63


on various sugars may indicate a failure of uptake, or a failure to express genes encoding

enzymes required for their utilization. The ability to utilize other alternative carbon sources such

as quinate and glycerol was only slightly affected by the acrB mutations, with the exception of

ethanol where lhe acrB mutants grew considerably less well than wild{ype (Figure 4.1G-H; Table

4.1).

Carbon catabolite repression oÍ alcAand amdS was assessed using plate assays (Figure

4.11-J), Test medium containing 2.5 mM or 10 mM allyl alcohol in the presence of glucose was

used to investigate the expression of the alcohol dehydrogenase encoded by alcA. The acrB

mutant strains appeared to be slightly more resistant than the wild{ype and creD14 strains,

indicating even tighter regulation of carbon catabolite repression of alcA in these mutant strains

compared to wild{ype. fhe acrB mutant strains were hypersensitive on medium containing

glucose plus 10 mg/ml fluoroacetamide, indicating an increased level of the acetamidase

expressed from the amdS gene compared to the wild-type strain, which is the opposite of the

phenotype conferred by the creD?4 mutation. The lack of growth of lhe acrB mutant strains on

medium containing glucose plus 10 mM acrylamide as a nitrogen source (Table 4.1) indicates

that this increased level of amdS expression is not due to constitutive expression, as acrylamide

is a substrate of the acetamidase but not an inducer ol amdS (Hynes and Pateman, 1970).

Fufiher, wild-type growth of the acrB mutant strains on acetamide as either a carbon, nitrogen or

carbon and nitrogen source indicates that the amdS gene is fully inducible (Table 4.1). Thus there

is a defect in the glucose-mediated repression oÍ amdS but no other effect on expression,

Growth on media containing the crl-amino acids 10 mM B-alanine, 10 mM GABA, or 10

mM pyrrolidinone as nitrogen sources was significantly decreased in lhe acrB mutant strains

compared to the wild-type strain, as was the case lor a creD34 mutant strain (Table 4,1).

Similarly, growth of the mutant acrB strains on media containing 50 mM GABA and 50 mM

pyrrolidinone acting as both a carbon and nitrogen source was also decreased compared to wild-

type and lhe creD34 mutant strain, but an effect was not observed on medium containing 50 mM

B-alanine. lnterestingly,lhe acrB mutant strains varied in their growth on medium containing 10

mM pyrrolidinone as a nitrogen source, with the acrBl5 strain having stronger growth, compared

to the intermediate growth ol lhe acrBl4 strain, and the weaker growth ol lhe acrB2 strain (Figure

4.1K; Table 4.1). This heterogeneity between alleles indicates that not all three alleles result in

complete loss of function,

Growth on media containing other amino acids as nitrogen sources, such as 10 mM

proline and 10 mM glutamate, was unaffected by the acrB mulalions. However, decreased

utilization of 50 mM proline and 50 mM glutamate when they were used as both carbon and

64


nitrogen sources was observed for the acrB mutant strains in comparison to the wild-type strain

(Table 4.1),

4.3 The acrBz mutation suppresses aspects of the creA, creB and creC

mutant phenotypes

The creD34 mutation was identified as a suppressor of the hypersensitivity to

fluoroacetamide conferred by the creC27 mutation. The effects of the creD34 mutation are

pleiotropic in that it also leads to suppression of the effects oÍ creC27 on other enzymes subject

to carbon catabolite repression, such as alcohol dehydrogenase l, but it does not suppress the

effects ol creC27 that are apparent in derepressing conditions, such as the poor growth on D-

quinate medium (Kelly and Hynes, 1977;Kelly,1980), The creD34 mutation not only suppresses

some of the phenotypes confened by the creC27 mutation, it also suppresses some of the

phenotypic effects of the creB1í mutation, and, weakly, of the creA204 mutation (Kelly and

Hynes, 1977;Kelly,19B0). These similarities between the phenotypes of creD and acrB mutant

strains led us to construct double mutant strains to test whether the effects of mutation in acrB

are also epistatic to some of the phenotypes conferred by creB and creC mutations.

The presence of the acrB2 mutation reversed the sensitivity confened by the creA204,

creB1937 and creC27 mutations to the presence of acriflavine in complete medium (Figure 4.28).

The creD34 acrB2 double mutant strain was even more resistant to the presence of acriflavine

than either of the individual mutant strains, indicating an additive effect, The resistance of the

creB1937 and creC27 strains to the presence of molybdate was reversed in the relevant acrB2

double mutant strains, and to a greater degree than in lhe creD34 double mutant strains (Figure

4.2D).

The acrB2 mutation results in reduced growth compared to wild-type on sole carbon

sources such as fructose, maltose, starch, cellobiose, ethanol and acetate (Figure 4.2E-J). This

reduced growth is also seen in lhe acrB2creB1937 and acrB2creC2T double mutants strains

compared to the single creB1937 and creC27 mutant phenotypes respectively, The creD34

mutation had no effect on growth of mutant strains on different sugars, and neither creD34 o¡

acrB2 mutations influenced the growth oÍ creA204 mutants on media containing alternate sole

carbon sources (Figure 4.28-J).

The inability of the creB1937 and creC27 mutant strains to utilize D-quinate as an

alternative carbon source was partially suppressed by the acrB2 mutation in the double mutant

strains, an effect not observed in the equivalent creD34 double mutant strains (Figure 4.2K).

65

Chapter 4: Molecular characterization and analysis of acrB

AComplete medium Complele medium + acriflavine Glucose + ammonium

*

t. *

.* *

D E

Glucose + ammonium + molybdale Cellobiose + ammonium Fructose + ammonium

* r,* n.

s

Gt**#G

Maltose + ammonium Starch + ammonium Acetate + ammonium

Figure 4.2 Epistatic effects of the acrB2 mutat¡on on creA, creB and creC mutantphenotypes. Strains indicated in (R) were grown at 37oC for 2-3 days on the following

med¡a: (A) Complete medium; (B) Complete medium plus 0.001% acriflavine; (C) 1% D-

glucose plus 10 mM ammonium L-tartrate; (D) 1% D-glucose plus 10 mM ammonium L-

tartrate plus 33 mM molybdate; (E) 1% D-cellobiose plus 10 mM ammonium L-tartrate; (F)

1% D-fructose p¡us 10 mM ammonium L-tartrate; (G) 1% D-maltose plus 10 mM

ammonium L-tartrate; (H) 1% D-starch plus 10 mM ammonium L-tartrate; (l) 1 .2% acetale

plus 10 mM ammonium L-tartrate,

66


ü

tü

+

J K LEthanol +ammonium Quinate + ammonium Glucose + pyrrolidinone

fs

t

M

Glucose + GABA Gluæse + fluoroacetamide Glucose + ammonium + starch

**irtu*

**t*

creA204 ueB1937 creC27 acrB2

creA204 creB1937 crej27 creD34acrB2 acrB2 ac¡82

creA204 creB1í qeà27 acrB2

creD34 crcD34 creD34 creD34

wild-type

P R

Glucose + âmmonium +

2.5mM allylalcoholGlucose + ammonium *10mM allylalcohol

Figure 4.2 Epistatic effects of the acrB2 mutation on creA, creB and cre0 mutantphenotypes (continued)... (J) 0,5% ethanol plus 10 mM ammonium L-tartrate; (K) 50

mM (-)-quinate plus 10 mM ammonium L-tartrate; (L) 10/o D-glucose plus 10 mM 2-

pyrrol¡dinone; (M) 1% D-glucose plus 10 mM GABA; (N) 1% D-glucose plus 10 mg/ml

fluoroacetamide; (O) 1% D-glucose plus 1% D-starch plus 10 mM ammonium L-tartrate

stained with iodine; (P) 1% D-glucose plus 10 mM ammonium L-tartrate plus 2.5 mM allyl

alcohol; (O) 1% D-glucose plus 10 mM ammonium L-tartrate plus 10 mM allylalcohol; (R)

Key to strain placement.

67


creB and creO mutant strains grow even more strongly than wild-type on medium

containing 10 mM pynolidinone as the nitrogen source, but in double mutant strains wilh acrB2

this phenotype is reversed, This is also seen to a lesser degree on glucose medium containing 10

mM GABA as the nitrogen source (Figure 4,21-M),

Carbon catabolite repression oÍ alcA, amdS and the (secreted) amylase genes were

assessed using plate assays (Figure 4,2N-O), fhe acrB mutant strains were as hypersensitive on

medium containing glucose plus 10 mg/ml fluoroacetamide as lhe creA204, creB1937 and

creC27 strains, as were the corresponding double mutants. Note that lhe creD34 mutation does

suppress the phenotype of the creBlS and creC27 mutant strains, highlighting again the

differences between the acrB2 and creDï4 mutant phenotypes.

Secreted amylase can be detected on a starch plate stained by iodine, with clearing of

the blue colour indicating the starch has been broken down. The creA204, creBlí and creC27

strains all inappropriately secrete amylase in the presence of glucose, which can be detected by

the size of the cleared halos, and the acrB2 mutation suppressed this phenotype (Figure 4.2O),

The acrB2 mutation reversed the toxic effects of allyl alcohol added to D-glucose medium

in creC27 and creB193Z backgrounds, the same phenotypic suppression previously seen for the

creD34 mutation except slightly stronger. The acrB2 mutation did not suppress the creA204

mutant phenotype of sensitivity to allyl alcohol in the presence of glucose.

4.4 Molecular analysis of acrB

The close genetic linkage oÍ acr? and creB (Clutterbuck, 1997) was taken advantage of

to clone the acrB gene. The BAC clone which contains creB, BAC 481 (Lockington and Kelly,

2001), was tested for complementation oÍthe acrB2 mutation in a transformation assay testing for

the reversal of the resistance to acriflavine in complete medium conferred by the acrB2 mutation.

Both the BAC 481 and the subclone p4AcrB, containing a 6 kb Clal fragment within the BAC 481

region, were found to complement the acrB2 mutation (J. Kelly and R. Lockington, unpubl. datal.

Thus acrB2 was located in the middle of the 6 kb insert of p4AcrB, and this plasmid provided the

starting point for the molecular analyses of the acrB gene presented here.

4.4.1 Sequence analysis of acrB

Since this work was undertaken before the A. nidulans genome sequence was publicly

available, the 6 kb insert of p4AcrB was sequenced using custom oligonucleotides (see Table

2.3), and AcrB is encoded within a 3111 bp sequence of the p4AcrB plasmid (Genbank

Accession No. 4F485329, Fig. 4.3), The acrB gene contained a single intron whose position was

68


CTTGACAGTAGCTGCCTGTAÀTGTCAGGCCATAÀÀGTTTCTGCCTTAGGCAÀTTTTGAGGATGTTTCCÀÀTCTTTAGGTCGTTTCGTCTTTTTCGÄÀCCAGGTCGTCTCTTCCTCTCCTTCTTCCCGCCTCTÀCACTCCCCCÀCGCCACGTCTTATCCCCTTCACTCTCCCC

*CTCfÀTCIT.A,TTÀTTCGAÀTTCTTCCCCTCTTTGCTCTGTGTATTCTCATATCTACTTGTTTCTTACGTTTCTCCCTTTTTGTCATTCATTCATTTTAACTGCAGAGACCCTGGCCTCGAÀTCCAGTCTTTTCCA,AÀCTCCCTTCCGCCCTTTCCCGCGÀÂCTTTTTTTGGAÀCTGCCATCGACTTCATTTCCACCTGGTCCAÄGTCCTTGGCTGCTATCCGCCAAGCCGÀCTCGGACCTGGGTGTTGGGTTCCCTCGTCCATGCCACGTTCATCGGCTÀCGGCTAGGAAGAGTCATAGCAATCGACÀÀGACCATGGCGGCGGCGGTTCTGGGAÄGA-AGCCAÀGCAÂÀ

MPRS SATARKSHSNRQDHGGGGSGKKPSKC,A.AÀAGTCAÄGTGGGCATTTÄÄÀCGCÀÀCATACÀ,ACGGTACTGCTGGATCCGAÀÀCTGGACCGTCTTCGCÀGGTCGACTGGCCATCG

QKS SGHLNATYNGTAGSETGPS SQVDWPSCATCGTTCTGGAGACCAÀTCGATCGCAGCAGCCGCAGCCÄÀÄTCTAACGGÀCCGGTCGATAGCTTGÀ.AÄGCGGACACCAÀCGGÀCGTHRS G DQ S IAAAÀAK SNG PVDS LKÄ DTNGRGGTTATCCGGGCGGATATGCGAAGGGAÀÀ.TGCAGACÀTGTCTTACGGGCAGÀCGAÀTGGTGGCGTGTCGCCGAATGGTGGACTCGCC

G Y P G G Y A KG NA D M S Y G Q TN G G V S P N G G I, AGGGCCGGCTTCACGTCGTACGGATAÀGTCGGTCACTGGGACCÀÀGAGGAC,AÀCTTCGAATGCGTCGGTGAÀTCCGTTCCAGCTGGCA

TCCACCATTCTCCGATCGTGTCCAÀTGTÀCGACACTATCGCCATCTTGATCTTTCTGCTTCAGCTCCCGCCTÀTGGTTCTCACTTTG

GTTCÀ.ATTCTTGTTTGCGTCCTTGÀCATTTATGCCTCCCAGCGGCACCGCTTCTGGATCCTTCACCTCCÄACTTCGATATTTTCCAGVQFLFÀSLTFMPPSGTÀSGSFTSNFDT FA

l1+11 =acrB1 4

GGACCCGCCGGA,\CCCCCTCGCTCGGTACCATGATTGCAÀTGGATGGTTTCTGCCTGCTTGTÀTGGGGCCTCTTTATGTGGACGTGGGPÀGTPSLGTM I AMDGFCLLVWGLFMWTWGCCCÀGAÄTTTTGCTCTCGATTTAGCCCATGTCCAGGTTGCCÀTCACTCTGGGCGGTGGAGGTGCAGGGÀÄ.AÀ.A.TGGTGGTGTCÀÀTAQNF ALDLAHVQVA I TLGGGGÀGKNGGVNGCGCTCTGCGTCGGTATTGTTCTGÀTTCTGCATCTCATACGCAGC.AÀÄGGA.A.TACÀGGATTTTGTCGTCGGCCATCTTGTTTCÀGCAALCVG IVL I LHL I R S KG I QDFVVGHLVSAÄÀÄÀTCATTAGCCCCGATTTACTGTCGCATTATTCTTACCTCATGCCCGCCGÄATTCÀAGCGCACCGÀATCGCAATCATCCCCGAGTKI I S PDLLSHYSYLMPAEFKRTESQSS P S

TGGATCCGGAGCCTGCTTGCTGTÀCATATTCTGGCCCAGGCGGGTACTGCGÀTGGCGAGGCGATCGATGACTAÄ.AÀÄTAGGACCCCGW I RS LLAVH T LAOAGTAMARRSMTKNRT P

GCCCCATCACGATCAGGCAÀÄ.CGCGTGGATACAGAÀGCGTCTGCCGGCTCAC,AÀÀCCCAGÀTCGACTCGGCGTTCGAÀTCCGCGGCCA P S R S GKRVDT EÀ. S ÀG S Q T Q I D SÀF E SAAÀGCGTTTCTTCCTATCTAGGCCCCGACGGGCAGÀTTATCACTGCCGCGCATAÀGGACGGCAGGGATCGTTTGATATCGGCAÀÀ.AÂ.4ÂSVS S YLG PDGQ I ITAAHKDGRDRL T SAKKCGACGÀAGGCAGGCA.AÄTCAAGTCÀGGAGCCGGCÀÀCCTTTTTGGGCTGCACTGGCAAGCACAAÀAGTCACGGTCATGAGGGAGTAT

G,AÄCATTCTAGGGCCTTGTCÀÀÄÀACTGCTAGAGGACTTGCTACGACGGAGGACGATCTTCÀÀGGCGTTTCTTTGGACGATGGACTT

GTTTGGATTACGTÀTGTGGATAGCTCGACGATTAÄGTTTGCAGCTGGGGATTTTGCGTCTTCGGACGACCATTCCGCGTCAGGTGTC

TGCGÀÂGCAGGCCGTGTGAGCAGCGAGGATGCGGAGCCGTTTTACGTCTGCGTCÀ¡.TGGTGCGCCATGGGCAÀCGGTGGTCÀTCACT

ÀAÄGAGCATGÀ.TCCTTCÄÀÄÀGCGTCTÄ.A.TACÂ.ATCTATTGGCGAGGCGAGATATCAGGTCTTGCACCCÀÄTTGCGCGTATACTTGCKEHDPSKASNT T YWRGEl SGLAPNCAYTC

\ l=acrB2TCTTTTGTTAÀATGCGATACGGATGAGGAÀÄTCTGCGCCATGÀGTGTCÀAGACCCCTGCGGCCAÄTGATGCAGAÀCAAGqTaaga IaS FVKC DTDEE T CAMSVKT PAANDAEQA

\-l =acrB75ttcg Ltc ta tggbttccga ttcg bc tcgatgtgg tc taat ttgtgcc ttccaLagCCÀÀTTCGGTGCCGGCCCCTCCGCAÀCCCTCA

NSVPAPPQPSTATCGACCATCCTCCCCAACAACCÀCGCTGAAGÄÀCTCGATCATCAÀTGCTGAGGCGA,CACTGAACGAÄÂÀGCGTGCTCGACTCCGAYRPS S PTTTLKNS I INAEAKLNEKRARLRÀAGGCCAAAA,ATGACCACA.A,GCTTGCTATTTCTÀÀGATCAÄGAÀGGAGCTGGACÀATTACACCAÄTCGTCTTCAGAGCGGCACGGATKAKNDHKLAT SK T KKELDNYTNRLQSGTDGÀÀÀ.ACAGGCAGAÄGCAÀCGCTCTCTTCÀÀTTGGAÃÀGGA-A.CATTCGACÀAACTGÀAGAGGCTACCGCCGCTCTGGACAÀCCAGATCENRQKQRSLQLERNIRQTEEATAALDNQ IGATAÀCTTGGGTAATGTTCCTGACGATGAGTATCAGGAGTGGGTTGÀÀCAGAÀGGCÀAÀGTACGAÀCGTGAÀTTGGAGCTCCTCAÀÂDNL GNVPDDEY Q EWVEQKÀKYEREL ELLKTCCGCCÀÀGGCAGAGATTGCTGCCACGCGTACCGCCAÀTGCTCGCGÀGTTATCTTCATTGGÀATCCGAGTTGA.A,CTCTACCACGCAÀS AKA E I ÀAT R TANARE I, S S L E S E LN S TT Q

CGGCGCGAÀCGTCTGCAGGGTCGCCGAÀCCAGAGTGAÄ,TGAGCAGTACGAÀCGGATCATCTCGGCCAÄCGCACAGGGTCTCÀATGAG

CGAGAGCGCCGCGCTGCAGAGCÀGTTTGCCCGGGAACAÄGATCAGTCGAAGTTGGAGCAAÀGTTTC,AÀCGAÀCÀ.A.TTCGCGAGCATC

AGTCAÀTCAGTGCAGGATTATCAGCTGCGCACCAGTCÀ.A.TTGTGGCÀÀCAGTGTACCGCCGTCGAÀCA,AGCCCTCCAGCÀGCAGTTG

CTCATGGAGCCCGCTCCGCTAÀCACCCGA.AGGCGÀGCTGCCCGGTACTAGTACGTTTGCCGÀCGCGCCCAGCGTGCCCTTGGGCACÀ

TTGGCGTCAA,ATATGCCAÄGCCACCGCTCGCTACTAGGACAGAGCTTTCCGCCGCTCÀÂGTCTAGTCCTCTGCAGCACTATGCTTCGLASNMPSHRS LLGQ S FP PLKSS PLQHYASCCÀATTGGÀACTGCTCCGTCTCATCCGACTAGTCCAÀTCGCCGCTCCATCCTACCAGCCTTTCTCCAGCTCGCCATTTGGTÄÀCGCGPTGTAPSHPTS P I AAPSYQ PFSSS PFGNAGCATCCTTCCTTGACCCGGACTTTGTCTACCGCGACCGTTCGTTCTCCAACCGCTCCGCACGCAGCAGCCTCTATGGCTCTGAGTTCAS F LD PDFVYRDRSF SNRSÀRS SLYG SEFCCGGACGCG,A.TÀACGGCCCGCCGTGTCCCCTTTGGCGTTGATCCTTTCGAGCTCGGTAÄCGAGAAÄCGACGCGGTTCGGGATCTGAC

l1881

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G PAS RRTDKSVTGTKRTT SNASVNPF QLA

ST I LRSC PMYDT IAl L IFLLQLPPMVLTL

R R R Q AN QVR S R Q P F VÌAA L À S T KVTVMR E Y

EH S RAL S KTARG LÀTTEDDLQGVS LDDG L

VWl TYVDSST IKFÀAGDFAS SDDHSASGV

C EAG RVS S EDAE P F YVCVNGAPWATVVI T

RRERLQGRRTRVNEQYERI I SANAQGLNE

RE RRAAE Q FARE Q D Q S KI, E O S FN E Q FA S I

S Q S VQ D Y Q L RT S Q L WA O C TAVE QA I, O A Q L

LME PAPLTPEGEL PGTSTFADAPSVPLGT

r7 40

PDA I TARRVP FGVD P FE LGNEKRRG S GS D

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2262

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AGCÀCCCCGCTCÀÀCGGTCCATCTGGCCTACGTCCTATCTCCAGTCCTTTCCAGCGAGCCGCCAGTCGCGCÀAGTGGAÀCCGGCAGT

STPLNGPSGLRPI S S PFORAASRASGTG S

GGCGGAÀGCGGAGGÀÀGTGGTAGTGGTAGTGGCAGTCCTAGCTCTGCCCGTGGA.AÀGGGA.AÀCTAÀÀCGGAACTGCTTGCCAGCCAGGGSGGSGSGSGSPSSÀRGKGN*CCAÀCTCAGCCGTCTGCAGTGACGACAAAÀGCATTGGAGATTGATCCTTACTCGCCTGCCCATATGTCCATATTTGGTCCTCGGÀCATGGCTCGGACTGTCCGTGTAGTGACTGGTTATATAGTTTGCCGTCGCCAÀCGGTCTATAGTATAGATCCTTGTGCTCGCCGCGTÀGAGCCACATCTTATATTTTGGCGAÀGÀÀÀÀÀGAG

Figure 4.3 Locat¡on ol the acrB mutations within the ac¡.B gene. The complete sequence of the acrB genomic region is shown (GenBank database Accession No

AF485329).Thes¡ngleintronof 63bpisdepictedinlowercaseleltersandlhe5'and3'splicejunclionandlarialsequencesareunderlined AputativeTATAboxandaputative CCAAT box are underlined and in bold lelters. The major stan po¡nt of transcription is indicated above the DNA by lhe # symbol. Bold italics indicate single

theoretical CreA and AreA binding sites in the 5' untranslated regions The amino ac¡d sequence is shown in single letler code beneath the DNA sequence, with

numbered amino acld residues indicated on the left hand side, and numbered nucleot¡de positions ¡ndicaled on the right hand side, with the f¡rsl nucleotide of the start

codonas+1 Thepositionandnatureof theDNAsequencechangesinlheacß2,acßl4andacr3l5allelesareshown,with(_)indicatingadelet¡onol lhebasedireclly below, and ( + ) ¡ndicaling an addition of a base al the position below


identified by sequence analysis of PCR amplified cDNAs, and the intronic sequence conforms to

the consensus 5' and 3' junction and lariat sequences for fungal introns (Gurr ef a/., 1987). To

determine the transcription stafi point (tsp) of the gene, 5'RACE (rapid amplification of cDNA

ends) of RNA purified from D-glucose grown mycelium was canied out using nested primers.

Direct sequencing of the nested PCR product and of several subclones of this product mapped a

single major lsp -221bp from the translation start (Figure 4.3). A TATA-like sequence is located

33 bp upstream of the tsp, and a CCAAT box 155bp upstream of the tsp. The acrB gene is not

present in the A. nidulans EST database (containing EST information for cDNA clones from a

mixed vegetative and 24hr asexual development culture ol A. nidulans strains FGSCA26

constructed in lambda Zap by Dr. R. Aramayo at Texas A and M University (USA)

http//www.qenome.ou.edu/aspero.html) indicating a low level of expression under these

conditions.

The acrB gene encodes a 1015 amino acid polypeptide. AcrB is predicted to contain

three trans-membrane domains near the N terminus, at amino acid residues 158-180 (l), 214-236

(ll) and 257-274 (lll), and a coiled-coil region, at amino acid residues 596-753 (SMART protein

motif analysis program, Schultz et a1.,1998) (Figure 4.4). The trans-membrane domain prediction

program, TMHMM2 (Krogh et a1.,2001), predicts the most probable location and orientation of

trans-membrane helices from sequence data, and this program indicated that the N{erminus of

the AcrB protein, and the region between the second and third trans-membrane domains are

most likely cytoplasmic, with the C{erminal region of the protein predicted to be external.

There is a hypothetical protein in contig 1168 of the TIGR Aspergillus fumigatus Genome

Database that shows 76% identity with the A. nidulans sequence, but these are very closely

related ascomycete species. Database searches (SwissProVSpTrEMBUPDB) using the BlastP

program (Altschul ef a/., 1990) revealed that the AcrB protein was not highly similar to any protein

in these databases. AcrB shows most sequence similarity with hypothetical proteins, one from N.

crassa, Q9C2R3 (31% identity over 1015 amino acids) and one from S. cerevisiae,YG2K(24%

identity over 861 amino acids). The hypothetical N. crassa protein contains three putative trans-

membrane domains and a coiled-coil domain, and although it shows low similarity along the

length of the protein with AcrB, specific regions such as the trans-membrane domains share

higher sequence identity (55% identity over 24 amino acids). The S. cerevisiae hypothetical

protein has a similar protein structure to AcrB, with three trans-membrane domains and a coiled-

coil region, but the sequence similarity is uniformly low throughout the protein.

70


acrïl4 acrB2 acrBl5* #

AcrB 1 1015

transmembrane

domainscoiled-coil region

Figure 4.4 Schematic of AcrB. The open boxes indicate the three transmembrane

domains, and the filled box indicates the coiled-coil region of AcrB. Asterisks mark the

location of the relevanl acrB mutations.

71


4.4.2 Molecular analysis of the acrB mutant alleles

Three mutant alleles of acrB were sequenced in order to identify functional regions of the

AcrB protein and to show that acrB ralher than a suppressor oÍ acrB2 had been isolated, We

used a PCR approach using primers spanning lhe acrB gene followed by direct sequencing of the

PCR products (see Table 2.3). Direct sequencing of acrBPCR products from wild{ype DNA was

undertaken for comparison. The acrB2 mutation is a single base pair deletion at nt +1734 at the

5' splice site of the only intron (Figures 4.3, 4.4). This would be predicted to disrupt the splicing of

the intron resulting in a frameshift after amino acid 577, and the additional residues,

GRYSFYGFRFVSMWSNLCLP, before terminating. fhe acrBl5 mutant allele contains a two

base pair deletion at nt +1815, which results in a frameshift after amino acid 584, with an

additional amino acid sequence of ATLISTILPNNHAEELDHQC before truncating. BoTh acrB2 and

acrBl5 gene products contain all three trans-membrane domains but lack the coiled-coil region.

The acrBl4 allele contains a C to T transition plus a one base pair insertion (T) at nt +627 that

results in a frameshift after amino acid 209 and truncates 84 amino acids later. The additional

amino acids are FARYH DCNGWFLPACMGPLYVDVGPEFCSRFSPCPGCHHSGRWRCREKWW

CQCALRRYCSDSASHTQQRNTGFCRRPSCFSKNH, The acrBl4 mutant gene product lacks two

of the three trans-membrane domains and the coiled-coil region and, since it is also recessive to

the wild{ype allele, probably represents a null allele.

4.5 Protein interactions ¡nvolving AcrB

4.5.1 Screening for proteins that interact with AcrB

The Stratagene BacterioMatch I Bacterial-2-hybrid system (B2H) was used to identify

proteins that interact with AcrB. The coding region ol acrB cDNA that incorporated the coiled-coil

region of AcrB (aa 582 - 1015) was ligated into the bait vector to produce the pBTccAcrB

construct. This vector was shown not to auto-activate and then used to screen an A. nidulans

cDNA library in the search for protein-protein interactions. There were fifty-five carbenicillin

resistant colonies that indicated an interaction may be occurring, but upon validation all proved to

be false positives. The A. nidulans cDNA library used in this screen was created by digesting a

previously made yeast-2-hybrid cDNA library with EcoBl and Xhol, and subcloning these

fragments into the target vector of the B2H system (R, Lockington, unpubl. dafa). Sequencing of

some of the false positives indicated that the pTRG-Library plasmids often had multiple partial

inserts that were out of frame, The availability of this cDNA library prompted its initial use, but

future work would require the construction of a B2H specific A. nidulans cDNA library,

72

Chapter 4: Molecular and analvsis ol acrB

4.5.2 Testing AcrB for interactions with other known proteins

To investigate whether AcrB homo-dimerises, the coiled-coil region ol acrB that was

ligated into the bait vector (see Section 4,5.1) was also incorporated into the target vector to

produce the construct pTRGccAcrB. No interaction was detected. The entire length of the creB

gene had previously been ligated into the target vector to produce the construct pTRG-BFLAG

(R, Lockington, unpubl. data). The pBTccAcrB vector was used to investigate a direct interaction

with CreB (pTRG-BFLAG), CreD (pTRGcreD, see Section 3.6.1), and ApyA (pTRGapyA, see

Section 3.6.1). The pTRGccAcrB vector was used to investigate a direct interaction with CreA

(pBTcreA, see Section 3.6,1) and HulA (pBThulA, see Section 3,6.1). The AcrB protein did not

interact with any of these known proteins in this system,

4.6 Discussion

Phenotypic analysis of the effects of three acrB alleles has shown that mutations in acrB

have a broader range of phenotypes than the resistance to toxic compounds such as acriflavine,

crystal violet, and malachite green previously described (Roper and Kafer, 1957) as they grow

poorly on a range of sole carbon sources including sugars, starch and ethanol, and they also

grow poorly on some cramino acids such as B-alanine, GABA and pyrrolidinone as either sole

carbon and nitrogen sources, or as nitrogen sources in the presence of D-glucose as a carbon

source. Genetic analysis has shown that the acrB2 mutation is a suppressor of the phenotypes

conferred by mutations in creB and creC. Strains containing ïhe creB1937 and creC2Z mutations

show derepressed expression of the a/cA gene, in that there is inappropriate expression in the

presence of D-glucose, and the presence of lhe acrB2 mutation completely reverses the effects

of these mutations as judged by plate testing. The reduced growth oÍlhe acrB2 containing strain

on medium containing ethanol as a sole carbon source may indicate that this is due to a failure to

induce alcA in either repressing or in derepressing conditions due to the loss ol acrB, and that

this failure in induction overrides the derepression caused by creA, creB and creC mutations. The

creB\937 and creC27 mutations lead to poor growth on D-quinate and D-glucuronate as sole

carbon sources, and the acrB2 mutation partially repairs this defect. These phenotypes are

consistent with a regulatory defect associated with the membrane and signalling, but not with a

simple interpretation that only permeases and transporters are affected, as in the case of ethanol,

a fat soluble compound, no permease is required or exists.

The amino acid sequence of AcrB indicates that the protein contains membrane

spanning domains, indicating the probability of its residing in the membrane. There is also a

coiled-coil region that would allow the prediction that the protein forms either a homo-dimer or

hetero-dimer. However, using the BacterioMatch I bacterial-2-hybrid system to directly test

73


whether the coiled-coil region is responsible for AcrB forming a homodimer suggested that the

coiled-coil region alone is not enough to form a homo-dimer. AcrB did not interact with CreA,

CreB, CreC, CreD, ApyA or HulA in the BacterioMatch I bacterial-2-hybrid system, but it would be

worthwhile validating these results in the newly released BacterioMatch ll bacterial-2-hybrid

system that is more sensitive and has a reduced background.

The striking aspect of the sequence is the lack of sequence similarity with proteins in

databases from other eukaryotes, including those for which the entire genome has been

sequenced, and it is clearly not a highly conserved homologue of any gene in a characterized

signalling pathway. lf functional homologues exist they are very diverged in sequence from AcrB

and thus unrecognizable. The CreB and CreC proteins are involved in a deubiquitination network

that removes ubiquitin from target proteins, and although these target proteins are not yet directly

identified, the mutant phenotypes indicate that they are proteins involved in carbon metabolism

and its regulation. Unlike AcrB, CreB and CreC are well conserved amongst most eukaryotes

other than yeast. The phenotypic evidence suggests that AcrB is involved in an opposite process

to deubiquitination, such as an ubiquitin ligase pathway, since a failure to add ubiquitin to

substrates could suppress the phenotypic effects of mutations that affect the removal of ubiquitin

moieties. Thus the ubiquitination/deubiquitination network in A. nidulansthat involves CreB, CreC

and AcrB provides an ideal genetic and molecular genetic system in which to unravel regulation

of protein stability.

74

Chapter 5: ldentification of an amvlase cluster

CHAPTER 5: IDENTIFICATION 0F AN AMYLASE CLUSTER

5.1 lntroduction

Alpha-amylases are enzymes involved in the breakdown of starch and maltose, and a

number of cx-amylase genes have been identified in Aspergilli species other than A. nidulans.

Shroff etal.(1997) observed an over-expression of secreted o-amylase activity in the presence

of glucose in creA mutant strains in comparison to the wild{ype strain, as indicated by a clear

halo on medium containing starch and glucose flooded with iodine, This suggested the presence

of secretable cr-amylase activity in A. nidulans that is carbon catabolite repressible. ln creA

mutant strains this o-amylase activity is derepressed, and inappropriately expressed at high

levels.

ln an efforl to identify o-amylase genes in A. nidulans, the A. oryzae Amy2 protein

sequence was used to identify sequences in the A. nidulans EST database that when translated

shared a high amount of sequence identity to Amy2 (R. Murphy, unpubl. dafa), This led to the

subsequent cloning and characterization of the cx-amylase amyB (GenBank Accession No.

AF208224). The EST database used to identify amyB was compiled by sequencing random

clones from a cDNA library made from mycelia grown in the presence of 1% glucose (carbon

repressing) and sodium nitrate as the nitrogen source (R. Aramayo, pers. comm.). That a number

ol amyB ESTs are present in this database implies lhal amyB is unlikely to be under the control of

carbon catabolite repression, AmyB lacked the typical fungal signal peptide found in the first 21

aa of most other identified cr-amylases, suggesting that AmyB is not secreted, Therefore the

derepressed, secretable cr-amylase activity found in lhe creA mutant strains was likely to be the

result of another cr-amylase in A. nidulans, and a second approach to identify cr-amylase genes

was adopted.

The cr-amylase gene from A. oryzae, amy2, was used as a probe in a low stringency

Southern of an A, nidulans À gDNA library. One positive À clone was identified, and designated

¡,OAMY1, which contained a 23 kb genomic Sac/ f ragment inserted into the Xhol site of IGEMl 1

(R. Murphy, unpubl. data).

This work preceded the public release of the A. nidulans genome database.

75


5.2 Sequence analysis of the amylase cluster

The l,OAMYl clone was used to create a number of subclones of this region, including

OAMYl R15,0SK+1/5, OAMY1Sac3.5SK+1 , OAMYl BamSK+1 , 0AMY1 BamO.7SK+1 , and

OAMYISacSK+1/3, which were then used to sequence the 10 kb region encoding the amylase

cluster using standard fonruard and reverse sequence primers, and a series of custom

oligonucleotides (Figure 5.1) (see Materials and Methods, Table 2.3), The amylase cluster

consists ol amy1, a putative transcriptional activator of the amylase genes, agdA, an

cx-glucosidase, and amyA, an o-amylase gene (Figures 5,1 and 5.2),

amy? encodes a662 aa putative transcriptional activator of the amylase (and maltose)

genes, and contains a Zn(llþCyso binuclear cluster DNA binding domain (pfam00172; aa 13 -53)

and a fungal specific transcription factor domain (pfam04082; aa 252 - 351), a region identified

by homology with a number of other fungal transcription factors including the transcriptional

activator XlnR, and the regulatory protein Gal4p from S. cerevisiae. The fungal Zn(ll)zOyso

binuclear cluster domain is a Cys-rich motif found in a number of fungal transcriptional regulatory

proteins that is involved in zinc-dependant binding of DNA, whereby two Zn atoms are bound by

six Cys residues (Pan and Coleman, 1990). The 5'upstream region ol amyR contains 5 putative

CreA binding sites, and thus may be under the control of carbon catabolite repression. AmyR

shares 69.8% aa identity with AmyR from A. otyzae, and 72.7% aa identity with AmyR from A.

niger.

amyA encodes a 498 aa cr-amylase enzyme with an oc-amylase catalytic domain

(pfam00128; aa28-382), and is classified as a member of family 13 of the glycosyl hydrolases, a

group of enzymes that hydrolyse the glycosidic bonds between carbohydrates. AmyA contains a

fungal cleavable signal peptide at aa 1 - 16 (von Heijne, 1986). The 5'upstream region of amyA

contains 5 putative CreA binding sites, and a CCAAT element that may be bound by the

AnCPiAnCF complex (see Section 1,4.3). No AmyR binding sites were found in the 5' non-coding

region oÍ amyA, in contrast to the promoters of the amylase genes found in A. oryzae and A.

nþer. No ESTs corresponding lo amyA were identified in the A. nidulans EST database

constructed from sequences obtained from RNA isolated from glucose-grown mycelia suggesting

that these CreA binding sites may be functional in carbon catabolite repression. AmyA shares

69J% aa identity with Amyl from A. lryaae,68,9% aa identity with AmyA from A. nþer, and only

61.2% aa identity with AmyB from A. nidulans.

AgdA is an o-glucosidase enzyme, 993 aa in length, encoded by agdA containing 4

exons and 3 introns in conserved positions in comparison to cr-glucosidases in other filamentous

76

Chapter 5: ldentification of an amylase cluster

SRI BS B RI SBB S

<-amyR

I oAMYlRrs.osK+1/5

\

agdA amyA

I ttOAMY1Bam0.7SK+1

L onl¡Ytsac3.ssK*1 J

L oAMYlBamsK*1 I

OAMYISacSK+1/3

1kb

Figure 5.1 Schematic of the amylase cluster, The shaded boxes represent the ÀGEM11

vector, with the lines in between representing the 23 kb gDNA insert; the double slash

indicates this region is not to scale. Relevant restriction sites are indicated above the gDNA

insert, with Rl = EcoRl, B = BamHl, S = Sac/, The arrows indicate the positions of the open

reading frames for amyR, agdA, and amyA. The bars underneath the gDNA insert

represent the different fragments that were subcloned and used as templates for

sequencing the region, The figure is roughly to scale as indicated by the scale bar,

77

Chaoter 5: ld of an amvlase cluster

l-79L57235313391

GAATTCAAGA.AÃÀTTAGTTGTGCAGTCCTTGTGAATTATGCGCACATGTATTCACCGAAGTACA.AAGCAGAGCTATAGAAGTATAAAGTÄA.A,TTATATTGTTCGTTCCAÀTGAGTCTATAGGTGGTATGTCATATCGTGTCATGTCAGCTGAGTCTCGTACTTCCTGGCAATAGGTATGAGGGTGGAGAGATAGTGATAATAAGGATGATAGATACATGGAGGGAGTAAAGACGTTGGGAAGATACGCAGATGTGCAAAACACAGTCCAATATCTCCGACAGGTTGATAGATATATAAAAACCTAGAATCCTCGGTAGCGATATATATACCCTTTCTTTGTATGGGCGAATTAGGGAGGAGAGCAGAAGGGTGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGTAGTGGTGGTGOTGGIÀGCCAGTCCAGAAGTCAAGTCAATAACCTCCCAGCTTGAAACGAGAG

*TI,I,RGAQFSI,TTCTGCAGCCATACTAGTGGACGAÄ,TGCATTCGCGAGACCGA.AAGGGGTACTGACGATGATCCAGACAATGAAGGCGG

EAAM S T S S HMR S V S L P VS S S c S IJ S P PCTGATCACTAGTCTGGTTCTGGTCGTTCGTATCACCAGAAGTTATTTCCCATGCACACGAATTTGTTGTCCTGCTGCC

QDSTQNQDNTDGSTI EWACSNTTRS GTGTGCTAGAATCCATAGTATTGTCATCAGCCGCGGGCCATGGTCCAGAGATATCTAAAGTTTCAGAAGCAATTGAÄGA

T S S DMTNDDAA PW P F S T D IJ T E S A I S SAGAGCTAGCGGAATTGAGAAGCGGAGGATTAATCTGGTGGA.AGTCACTGATAGAAAGCGGCGTCGGGGAÄTCAAAGCC..S S A S N I, I, P P N I Q H F D S I S L P T P S D F GGAGGGTGCCTTTGCAGCGCTCAAGGAGAGATGGGAAGAGGTACGACTGGGAGCCACGGATGCGGGAGAGGGTGGAÃAG

IJ I G K C R E Ir L S P F L Y S Q S G R I R S L T S LGATACCCCAGAGGAGCTCGCGGGGGTCGACGGTTGACTCGGCGAGGCTGTTTGCAGCTTTTGTGGAGAGAGAACGTGA

I G I{ IJ TJ E R P D V T S E A I, S N A A K T S IJ S R SGACGTCGGCGATAGATGTACCAAGATCGAAGAGCTTTTGTTCctgcataatcgtcagccttaatcgaagtaaattgctVDAISTGI,DFLKQE

aaagtggtsgaagactacCATCCCAATCCCATGCGCGTCGACAGCCCCCTGCGATGCCTCCGCAATGACAGACATAACAMGTGHADVAGQSAEAIVSMV

GCCTTGCCCACCATTACAGGAAGATGGAACGGGAGGACCGTCTCATTCCGCGAGCCTGGCTGCGATGCACGCGTCATGA K G V M V P IJ H F P IJ V T E N R S G P Q S A R T M

GAGAGTTTCCACATCATGGCCTGAAGCCACTGCTGTGTGATGAGGATATCAACCTTTTGGATTTCCGAGACACCCTCG

ATGAAGCCGTAGGCGAGAATCGGGTCGTCGGAGCACAGGACTTGCGGTTTGTGGATGGAGTTGCGGAGCATGACAGGT

r F G y A rJ r 't_p_"__"_-c:_":J:Þ_!:E:E:iI:J'_-.:_":y:!:"_¡TTCGCTTGTTGGAGGGCATAGCCTctgcagcacttgE cagt t t aEggt ctatt cagctctacggaggt aaatacCGTTKAAAI,AYG l-o_ a

r-"- J

469

547

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553

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359

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S L KWM MA Q IJW O O T I I, I DV K Q I E S V G E

IJ S I P K S IJ TA Q I A S T P P M E S S G D R N N G

G G S V S G G A S V W D Y IJ N V S I' S E F V G I IJ N

LCTGTGATGAAGAGCAGCCAGAAGACCCGTCGCTTCTGTTCTGCTTCTTCGGTGTTCAÀCTCGGCGTATGTTGATTCGCIE T I F IJ I, W F V R R K Q E A E E T N I, E A Y T S E I

êeAõcL[etc-c-a.n]rõcêùãelcõeir-e-nrAicËc-cic-eõüÃcãr1,c-¡ãc-cÃrîcõclc-e-rc?ie-cc-e-cr-cõa?Ãrîfc'

AR VA E A I, I, E E G S M S S N E GA Q F H S P D A

F- 5_ _t _ g _L_ _L _L _v_ lvr_ _s_ 3 - å -e, -c-CGTATGAAGCAAATAGGÀAGAAÎGAGGTTAGAAGACTCTCAACATTCATATCCTCAATTGGGTCGCAATCTTTCCTTGb-1-Ë-Ã-r--f, -r--il-s-ï--r,-t-5-d'-\¡-r,iî--D--E-1 p D c D K Rt-r-

----JCGCGCACGGCTTCGGCGAGCAGTTCTTCACCGGACATTGATGAGTTCTCACCGGCCTGAA.AATGCGAAGGATCCGCGA

CGGGCGTTGCTCCGTCGAGTTTCAGCTGGATATGTGTGGCGGCACATAGGGAGGCCAGAAAGGCGTACCGCTGGGGAGV P T A G D IJ K L Q I H T AA C I' S A IJ F A Y R O PTTAGACGCTCTGGATGGTGGCAGTCCTGCTGGAGCTCTTCTCTTCGGACCACGGGCATGATAGGAAACATATATTTCAT L R E PH H CD Q Q IJ E E RRVV P M I P F MY KAGTAGACATTGACGTGCGCCAGAAGCACAGGTGCGGTCAGAATCCGCGGCGAACGAAACAGCACCGCAGTCGGAGAGT

CCGATAGGGAGTTCAGAGAATCCGGCGAAGAGACGAGTTCTGGTGGAGGCAGCCGGCTGAATGAGTCAGGGTGTTCAGD S I, S N IJ S D P S S V I' E P P P IJ R S F S D P H EGCGGCAGATATTGCGGCTCTTCCATGÄAGGGCGGAGACGTGGGCGAGCCGCCGACCGGGTAGCTCGTAGGCTCTGCATP P IJYQ P E E MF P P S T P S GGV P Y S T P EAACCATTCCCTCTCTGCAGAGAATCGTGGAGGATCATACTGGAGAATCTGTCGGTCTCTGGCCACTAGTGGATGGATAGYW E R E A S F R P P D Y Q I, I Q R D RAV I, P H IGCGCTAGAGGGTATAGAGTCCGA.AATTTCGGCCCTTTGCGACGCAGCACATCGCTGTAGGAGCAGGAAAGGAGCAGGC

L Y V N V H A IJ I' V P A T I' I R P S R F I' V A T P S

PAIJPYIJTRF K P G K R R IJ V D S Y S C S I' IJ I'GCTGGCACTTGTCGCACGGGAGCTCTCGTGAGCACTTGATTTTCCTCCGGCGACAGTTGTCACAGGCCTGCTTGAAAG

FAIKRRR ND

PKHKPAAPSAM25752653273L2809288729653043312r3L99321733s5

AGATGAGAAGCTAGTCTTGAÃGAGGCATGCGGGTCATGTCGGATTGCTCGTCAACAGTATAAAATCGCTATAGTCCTTGCAGACGTACGCATGGGTGAAAATCTGGiGGAAAGCGAGCOGG'OGAGGGAGAGGATAAAGAGAGACGGGCAGGAGTAÀTCGGATAGCAGTGACACTAACTCCACTATCCGCTTGCCCTCCGTATAGTACTCCGTAGTGGCCTGCAGCCGTAGTCAGGCTCAGCCCCGCGTTACACCATTGCTTCTCGGCTTCGTCTCTTAGCAACCCTTAACAGTTCATTTAAGGCTTCTTCGCGTTAGCTTTGCCGGTGGACTGCACTCCAGCCTGCCACCTTTTGCCCAGAGAAAAGAGCGAGGTGACGGCTACAGCCAGGCCCGAGAGCCGGCTGTCAGAAACCCCTGAATCCTGGATCCGGGATTGTTGCCCAGTATGACGTCTTGGCAGCATGCGCGATTTTCTAGAAACCTCCCCTGCATGAAGCCCATTTGCCTGTGTGATCCTTGGTCTAGCGGTCGTCCCCTGCGCTGGCCATTCGTCAGTGTCGAGTAAGACATGÄACGCCTCTTTAGA.AATCGGGCTTCTGTGGCTCACTTAAGGGCCCAAGTCTGTCGGGAAGCCGAGGAACAGCAGCATGACCCAAGGAACCGATGCAGTGCCA.ACCACAGATGCCGAATCCTCGTACATCGTCTTGGTGGTGTTGATGTGCAGCGACCGTTCTCCACCGTGGGTTCTGGGAGGGGGCCCGATTCCAGTCTCAATCTCGAGGTCTGTTTGAGTTTTAGGATAACGAAGCGCTGGATGTCTGGATTTCATGATTTCATGTGATACTCATGGTGCCGAGA

78


GCATAGCTAGCAGTCCAGACTCCGG¡ATTATCCAGGAAATCGATCAAGTCGACTGTTCTCAGTCTGGTCGGGGACTGGGOTTAGCGGGAAGGCTOGAG¡ACTGGAGTTGCTCACCGGTGGTAATTTTAGGCATGGCACGCCCGTAACATTACGCACACAÀTACGGTATTCTGGIGGATGAACCCTGCTCTGGGTAGATACTCCGTACAGAATAGCTTGGCTGAGAGTTAACACTCCACAATCTCCTCTCTTCATCCTCTTCCATCACCCGGTGCTTCGACCTTCGTATCCTGCATCCTTGATATCAACTATCCTCGACTATAACAGCTTCGTTATAGGCT GATATG CAGCATTTTGCAGTGCCTCCACCCCTccalcccATcrcAcrcccAlccrrr dqrisaoeccecrccrccccocATcAcccAcrcccrrccrGCCTGCTGGCTTCTGGCTTCAÀCATGGGCTCTCTCTTCTGTTCATCCTCATCATACGCCCCTTGCATGCATCGTTGCGTCTAGGATTCTAA.AGTCAAATGGATGATCGCCGCGGGACGTGCCACAGCATGGTTTGACTCTTGCTTGTCTTGTGAATCCGTGATTTTCCCGTCGAGTTCGAAÀCCTCGAAGATTAAATTGAGTCAAGGCCGTGGTTGTTGGATGATCTGATCCATCGTACGATTGGAAAAGCCTCCTATCCCCACTATGGTCCGCTlCCTGCACCTAGCTGGGACCCTTCCGGTCCTAGCCTC

TGGCGCAGTCCAGGACGCCCTCCGGCCAGTTGCCGAGTCCGCCGCGACCGTAACCGCAACCGCGACCGTAGCCGGGCA

ACAGGCTCAATTTACACTCTCGGATTA TTGCCAATGTGGACGATCCCGAAGCCGT

ATTCATCGAGTTTGTGACATCTTTGCCGGAGGAATATAACCTGTATGGTCTAGGAGAACGCATCAACCAGCTCCGTCT

GTTGCGAAACGCCACACTGACCTCCTATGCGGCTGACATTGGCAATCCGATTGATGCgT

cttgccccatgct

acgttcggttactctcat c

TACTTCTCAGTTG

34333s1135893667374538233901397940574135

4213

429L

4369

4447

4525

4603

468r

4759

4837

4915

4993

50?1

5L49

5227

53 0s

53 83

5461

s539

5617

5695

5773

585r_

5929

6007

608s

6r63

624r

6319

6397

647 5

5553

TTGCGAATTTCGAGA.AATTTGAGATTCCGTTGGAATATCTCTG9T at gcacgaatacgcacctatt Egaat ccE agct

AGAAATTTTGAA.AATGACGAGTATAGATTTCCATATAACtacaÈctt

16

42

58

84

110

r.3 6

t62

188

2!4

233

25r

277

303

329

355

369

39L

4]-7

429

455

4 81-

507

533

559

585

6rr

637

663

689

775

74L

767

CCTAATCCGCAAAACGCTTCAGATTCgT aagttt Et cgEÈtt t at tgc caagt actgctgacaat ct t.agTTATGAAA

TGTTCGGAGTCG

79


GGTACGATTGGTACACACAAACTGCAGTTGACGCCCAGCCGGGAGTCAACACGACGA TCGACGCGCCCCTCGGCCATA

CGTGGGCACTGTTAGTCGCACTCGGTAAAGACGGAACAGCTTCAGGCCA TCTATACCTCGATGATGGGGAGAGTATCC

AAGCCAACCCA

793

819

445

87r

897

923

949

975

983

TGGTGCTTGGG

TACTTGGGATGTTCCCCGICA

CTCÎGCCGGCTTGCAAGGTTCTCCACTGCACCCCTGGTAATCTCCTGGCGTCGTGGTTGGACTGGCGTGGCCATTCTTTGCCAACCATTGCAGAAACCTCCGACAAAGCCGGATACTTGGGGGATGCTCAGCTGGTCGTAGGATACATGTGATAAATATGGGCACCCCACCGTAGTGTTAGGTGTTGTAGAGTGTAGACCATCAACAAGATCCTTCTGCAACGATGCGGTCGCC

CACCGACCGGTTTGCCCGCACCGACAACTCGACAACCGCCGAATGTGA ctggacct

agattttgatt cËaaEt

6631

67 09

6787

686s

6943

702L

7 099

71_77

7255

733374!L7 4897567

'7645

7723

7801

7879

7 957

8035

I 113

8191

8269

8341

8425

8503

85 81

8659

8737

I 815

8893

8971

9049

9127

9205

9283

936r-

9439

GGACTACATCCAGGGGA

GGAGGCATACCA

GACTACTCTAgtaaagc

ACTATCCCATgtacttt cccactatt

cggttcttctaggcaatgccacEgaccagacaat

ccttgagtaatggggtt.t

aacgataagtgc

tgct t t ts at agat gc caa! ac

ccctcccctatcctscccagtactgtatacatatctatgaaggtgf tct cacat ccaaaaca

4

30

49

63

89

100

125

136

L57

183

209

2r3

238

264

272

298

3L6

329

355

381

396

405

43t

451

483

tatcccgattacctgcatgcccgctaacÈgtacca9

tscct caacccaattt ccc

atctctctcctt.tcccctacataccgcggcc

80


95]-7 GGGAGCGGGATTTGCGGCCTGTAGTCGTACCCGATGATGAAACATAATGGACAAGTTGGTGAAAGGTTGGGCCAGAGG49r

9s 959673975L

TTGGACGGTCGTGAGGGTTATATTGGGGACGGTGAATATATATAGGAACATCGTTCCTTATAGTGCGGATGGTGTGGTAAGTATAGACTTTGGATGAAATTCGTTTTTGGATAATTGTGTCAGTATATGATTTGGTGTGGTCTTTTCGTTTGATTTTAGCATA

Figure 5.2 Nucleotide sequence of the amylase cluster, The amino acid sequences of AmyR,

AgdA and AmyA are shown in single letter code beneath the DNA sequence, with numbered

amino acid residues indicated on the right-hand side, and numbered nucleotide positions

ind cated on the left-hand side, The peptide encoded by amyR is highlighted tn ye he

by ispeptide encoded by

in

is highlighted in , and the peptide encoded

high ighted lntrons are in lower case letters, Consensus CreA

are ind icated by bold-face type, elements are highlighted in , and the

in the agdA promoter are grey, The Zn(ll)zCyso b nuclear DNA binding doma n

of AmyR is boxed with a

boxed with a botted linei.

and the fungal specific transcription factor of AmyR is

Sequences were deposited in Genbank with Accession numbers as follows

Amylase cluster nucleotide sequence 4F208225AmyA protein sequence 4AF17103.1

AgdA protein sequence A F17102.1

AmyR protein sequence 4AF17101.1

tinuous I

B1


fungi, AgdA contains a glycosyl hydrolase family 31 domain (pfam01055; aa 578-953), another

member of the glycosyl hydrolases that hydrolyse glycosidic bonds, The 5' region of agdA

contains 11 putative CreA binding sites, 2 putative AmyR binding sites of the (5'-CGG-No-CGG-

3') type, and a CCMT element that may be the target of the AnCP/AnCF, AgdA shares 73.6% aa

identity with AgdA from A, oryzae, and72,40/o aa identity with AglA from A. niger.

5.3 amyB is not part of the amylase cluster

To determine whether the previously characterized amyB gene resides within the

amylase cluster region, a BAC library was screened to identify whether a member of the amylase

cluster and amyB colocalized to a single BAC clone (which typically contain between 50-150 kb

gDNA), An A. nidulans BAC library was screened using amyA and then amyB as a probe. amyA

hybridised to 33 BAC clones, and amyB hybridised lo 24 BAC clones. No overlapping

hybridisation was seen. Therefore, amyB cannot be within approximately 150 kb of amyA and

does constitute part of the amylase cluster containing amyA.

Verification of these results came with the public release of the A. nidulans genome in

March 2003. amyB resides on contig 1.55, linkage group Vl, and the amylase clustercontaining

amyA on contig 1.32, linkage group Vll.

5.4 Regulation of the amylases

To determine the transcriptional regulation of the amylases identified in A. nidulans,

Northern analyses were undertaken. RNA was prepared from wild-type and creA204 mutant

strains under various growth conditions, including overnight (16 hr) incubation in media containing

as a carbon souÍce; 0.1% fructose, 1% glucose, 1% starch, 1% maltose, and also 0,1% fructose

for 16 hrs followed by 10/o starch for 4 hrs, 0,1% fructose for 16 hrs followed by 10/o maltose for 4

hrs, and 1% glucose for 16 hrs followed by 10/o starch for 4 hrs. RNA gel electrophoresis and

Northern hybridisations were performed using two different methods (see Section 2.3,7), but

under no experimental condition was any hybridisation detected when amyA, amyB, or amyR

radiolabelled DNA or RNA probes were used (see Table 2,3), indicating low levels of transcript

under the conditions tested.

ln order to increase the sensitivity of the assays, RNase protection assays (RPA) were

performed on the same RNA samples used in the Northern analysis, using amyA, amyB and

amyR as probes. Again, no transcripts were detected under these conditions.

82

Chaoter 5: ldentification of an amylase cluster

5.4.1 Alpha-amylase secretion plate tests

The absence of detectable transcripts in Northern and RPA experiments suggested that

conditions considered to induce c-amylase activity, i.e. the presence of starch/maltose, were not

inducing transcription of the amylolytic genes to detectable levels, Simple secretion plate tests

were performed to identify conditions that resulted in secreted cr-amylase activity. By spotting 20

pl of an overnight A. nidulans culture onto a 0,5% starch plate, incubating the plate at 37"C (for 1

hr, 4 hrs, and 16 hrs), and then flooding the plate with iodine, the amount of cr-amylase activity

can be determined by the size of the cleared halo (see Section 2.3.11).

For the wild-type strain, the only condition in which secreted amylase activity was

detected was in mycelium grown in medium containing starch as the sole carbon source (Figure

5.3). ln a strain containing lhe creA204 mutation, secreted amylase activity was detected in all

conditions containing either starch or maltose. These results are consistent with the presence of

secretable a-amylase activity under the control of CreA,

5.5 Discussion

The amylase cluster in A. nidulans consists of the transcriptional regulator AmyR, an cx,-

glucosidase AgdA, and an a-amylase AmyA. This is the same arrangement of genes as

observed in A. oryzae (Gomi et a/., 2000), The sequence of all three components of the amylase

cluster was submitted to GenBankon24lll/1999 (Accession No, 4F208225), Tani et al. (2001a)

independently reported on the cloning and characterization of the amyR gene from A. nidulans,

and had submitted the sequence of the amyR gene only to GenBank on 3/6/99 (Accession No.

48024615). Tanief al, (2001a) designed oligonucleotides to the 5'- and 3'- ends of lhe A. oryzae

amyR gene, and then PCR amplified amyR using A, oryzae gDNA as a template, The zinc

binuclear cluster domain region of the A.oryzae amyR PCR product was used in Southern

analyses to probe an A. nidulans gDNA library, and a 12 kb EcoR/ fragment containing amyR and

the 5'-part of the agdA gene was identified and sequenced, The two AmyR sequences entered

into GenBank for A. nidulans are identical.

Amylase secretion plate tests indicate that there is secretable a-amylase activity in the

wild-type strain when grown for 1ô hrs in medium containing 1% starch, but RNA isolated from

wild-type strains grown in this condition lacked detectable levels of amyA and amyR transcripts.

The 4 hr induction period by starch/maltose did not result in observable amylase activity by

secretion plate tests. Preliminary Northern and RPA investigations into the regulation of the

83


wild-type creA204b

fructose

glucose

starch

maltose

fructose;+4hrstarch

glucose;+4hrstarch

fructose;+4 hrmaltose

glucose + starch

glucose + maltose

Figure 5.3 Amylase act¡v¡ty secret¡on plate tests. 20pl of overnight culture was

spotted onto a 0.5% starch plate and stained with iodine after a 16 hr incubation at

37"C. Clearing indicates starch breakdown, and therefore amylase activity, Growthconditions were, in order from top to bottom ,0.1% fructose, 1% glucose, 1% slarch, 10/o

maltose, 0.1% fructose for 16 hrs followed by 1o/o starch for4 hrs, 1% glucose for 16 hrs

followed by 10/o starch for 4 hrs, 0,1% fructose for 16 hrs followed by 10/o maltose for 4hrs, 1% glucose and 1% starch, 1% glucose and 1o/o maltose, All cultures were grown

for 16 hours (unless stated otherwise) at 37"C, with ammonium tartrate as a nitrogen

s0urce,

84


amylase cluster had revealed that the level of amyR and amyA transcripts were below the level of

detection for all of the conditions tested in both wild{ype and creA204 strains.

Tani et al. (2001a) disrupted lhe amy1 gene, and these disruptant strains grew poorly on

media containing starch, maltose and iso-maltose, while growth on glucose, fructose, cellobiose

and a number of other carbon sources were not affected, suggesting that only amylolytic enzyme

expression was affected. The effect of lhe amy? disruption was also examined at the

transcriptional level, using taaG2 from A. oryzae as a reporter gene since A. nidulans produces

such low amounts of amylase, leading to difficulties in detection of amylase activity and mRNA

(Chikamatsu ef a/., 1999; Tani ef a1.,2001a).fhe amy? disruptant strains showed no detectable

amylase activity, and the taaG2 gene was not transcribed in the absence of AmyR in comparison

to a wild{ype (amy4 ) strain carryinglhe taaG2 gene that was transcribed at detectable levels by

Northern analysis when grown for 36 hrs in medium containing 2% starch plus 2% glycerol. Tani

et al. (2001a) determinedlhal amy4 expression was regulated by carbon sources, with the most

abundant amy1 transcription observed when grown on starch, followed by maltose, then glucose

and glycerol as determined by Northern analysis, This is in contrast lo amy4 transcription levels

in A. orzyae which were equal in maltose- and glucose-grown conditions (Gomi etal.,2000). As

in A. oryzae, amy? expression in A. nidulans is negatively controlled by CreA (Tani et a\.,2001a),

The inability to detect amyA lranscripts in Northern and RPA experiments carried out in

fhis project is consistent with the difficulties many other groups have faced when studying

amylases in A. nidulans, which is why [he taaG2 gene from A. oryzae has often been used as an

amylase reporter gene, as noted above. The growth conditions which resulted in detectable

amyHtranscript by Tani ef a/. (2001a) included a25hr incubation at 37"C in media containing 2%

carbon source (eg. starch), compared to the 16 hr incubation in 1% starch carried out in this

project. Recently it was shown that isomaltose, converted from maltose by a-glucosidases, is

the most effective inducer for amylase synthesis in A. nidulans (Kato et a1.,2002b).

The lack of an AmyR binding site in the promoter region oÍ amyA is consistent with the

low levels of amylase expression seen in A. nidulans. As previously mentioned, an amylase

cluster also exists in A. oryzae (GenBank Accession No.48021876) with a similar arrangement

of genes to the amylase cluster identified here. However, the region between the stop codon of

AgdA, and the starl codon of AmyA in A. oryzae is significantly longer than in A. nidulans (2447

bp compared to 388 bp respectively), and there is an AmyR binding site in the promoter region (nt

-261)for amyAin A. oryzae that is missing in A. nidulans, indicating that the A. oryzae amylase

can be induced by the pathway specific regulator AmyR whereas lhe A. nidulans amyA cannot,

85


Ihe amy4 disruptant strains showed a similar mutant phenotype to the malAl mutant

strain of A, nidulans (Roberts, 1963), with poor growth on starch and maltose, and Tani et a/.

(2001a) showed that the genetically identified amy1 and malA are the same gene.

The S. cerevisiae transcriptional activator for genes involved in maltose utilization,

Mal63p, was aligned with AmyR and five homologous regions were identified [Zn (aa 13-54),

MH1 (aa 152-2141, MH2 (aa 234-375), MH3 (aa 419-496) and MH4 (aa516-542)l (Tani ef a/.,

2001a), The MH2 region of AmyR is required for the transcriptional activation of the amylase

genes, and a truncated AmyR protein that lacked the MH3 and MH4 regions functioned as a

constitutive activator, implying this region may be involved in negatively regulating transcription

(Tsukagoshi et al., 2001).

Two AmyR binding sites in A. oryzae were identified as (5'-CGG-No-CGG-3') and (5'-

CGGAAATTTAA-3') (Petersen et al., 1999), and presumably these sites will be recognised by

AmyR in A. nidulans, Tani et al. (2001b) identified one putative AmyR binding site in lhe agdA

promoter nt -396 to -383 (where the translation start site is +1), and showed that this AmyR

binding site is capable of induction, and that AmyR binds to this region. Most Zn(ll)2Cys6

binuclear cluster containing proteins bind as homodimers to the CGG repeats of the binding site,

and mutational analyses showed that both CGG triplets in the agdA promoter AmyR binding site

are required for high level induction by AmyR (Tani et al., 2001b\. Tani ef al. (2001b) failed to

recognise a second AmyR binding site in the agdApromoter, located at nt -303 to -316, but their

results are not compromised by this as the experiments pedormed did not include this second

AmyR binding site,

With the public release of the A. nidulans genome in 2003, it made broad searches of the

genome with specific domains possible. The glycosyl hydrolase 31 domain of AgdA is found

within the genome an additionalfive times, including AgdB (Kato et a1.,2002a). AmyA contains an

cr-amylase domain, and this domain is shared with AmyB and seven hypothetical proteins. lt is

interesting to note that two of these hypothetical proteins, 4N3309.2 and 4N3308.2, physically lie

adjacent to each other in the genome. None of the proteins containing an o(-amylase domain

also contain a starch-binding domain, except for AmyB. Three hypothetical proteins were

identified in addition to AmyB that contain the starch-binding domain. AmyR contains both a

Zn(ll)z0yso domain and a fungal specific transcription factor domain, both common domains

within the genome (123 and 110 examples respectively, with a proportion of these containing

both, such as FacB).

86


The amylase cluster identified here contains the transcriptional activator AmyR that has

been more thoroughly worked on by Tani et al. (2001a, 2001b). Preliminary investigations into

amylase regulation had begun when the regulation oflby amy? was described by Tani ef a/,,

(2001a), and as such no further work on the amylase cluster was pedormed. The presence of a

number of additional amylolytic proteins to those already characterized, based on genome

sequence searches, leaves room for the characterization of these amylolytic genes and whether

they are functional in A. nidulans.

87


8B

Chapter 6: Conclusions

CHAPTER 6: CONCLUSIONS

ln the unicellular eukaryote S, cereyisiae, carbon catabolite repression involves the DNA

binding protein Miglp (Nehlin and Ronne, 1990), which recruits the Ssn6p-Tup1p general

corepressor complex to effect repression (Tzamarias and Struhl, 1995), Miglp nuclear

localization is regulated bythe protein kinase Snf1p, which phosphorylates Miglp in the absence

of glucose to trigger nuclear exporl of Miglp and relieves repression (Devit et al.,1997\. Miglp

shares a high degree of sequence similarity in its DNA binding domain with CreA, the master

DNA-binding repressor required for carbon catabolite repression in A. nidulans. However, the

mechanism of carbon catabolite repression in the multicellular eukaryote A. nidulans is quite

different, as CreA does not appear to recruit a general corepressor complex, and the Tuplp

homologue in A. nidulans, RcoA, is only weakly involved in carbon catabolite repression (Hicks et

a1.,2001), and no highly conserved sequence to Ssn6p is found in the A. nidulans genome,

Lockington and Kelly (2002) proposed a model for carbon catabolite repression in A.

nidulans that includes the transcriptional repressor CreA and a deubiquitinating enzyme complex

comprising at least CreB and CreC that are involved in a regulatory de/ubiquitination network, ln

this model, the CreB/CreC complex acts directly on CreA to affect the stability or activity of CreA,

and thus also effects carbon catabolite repression. ln the presence of glucose, the coiled-coil

region of the CreB deubiquitinating enzyme recognises ubiquitinated CreA as a substrate, and

cleaves the ubiquitin chains from CreA. This results in CreA no longer being targeted for

degradation via the 265 proteasome or having its function altered, leaving CreA active to bind to

the promoter of glucose-repressible genes and effect repression. ln the absence of glucose, the

levels of CreB are reduced via PEST-mediated degradation of CreB, resulting in CreA remaining

ubiquitinated, leading to the degradation of CreA or an alteration of its activity, such that carbon

catabolite repression is relieved. Consistent with this, creA mRNA levels do not directly correlate

with CreA-mediated repression, implying that protein modification andior stability of CreA could

be an important component of the carbon catabolite repression mechanism (Arst ef a/., 1990;

Shroff et al., 1996; Strauss et al., 1999). ln addition, Strauss ef a/. (1999) noted that CreA

contained a sequence similar to a consensus sequence that triggers ubiquitination in yeast,

implying that CreA could be modified by ubiquitination in the absence of glucose, resulting in

inactivity/degradation of CreA,

The CreB/CreC complex also plays a role in the turnover of some enzymes and

permeases (such as those in the proline and quinate utilization pathways) in both the presence

and absence of carbon catabolite repression, as creB or creC mutations lead to the failure to

89


express enzymes for the utilization of L-proline (as an example) in both the presence and

absence of glucose (Lockington and Kelly, 2001). Regulation of the proline utilization cluster

would suggest that this action is likely to be directly on the permeases or enzymes themselves,

rather than via the pathway specific regulatory proteins involved in induction, as the proline

permease PrnB is directly regulated by CreA (Cubero et a1.,2000), whereas the pathway specific

transcriptional activator PrnA has constitutive nuclear localisation and is not dependant on proline

induction (Pokorska et al., 2000). The quinate permease QutD is another likely target for the

CreBiCreC complex, and analysing the stability of these permeases, and whether they are

ubiquitinated in vivo could help define the role for CreB/ CreC in their regulation.

The CreB/CreC regulatory deubiquitination complex may not act via CreA to affect

carbon catabolite repression, but could directly act to stabilize permeases, transporlers or

glucose sensing proteins by the removal of ubiquitin, thereby altering the cellular concentration or

localisation of signalling molecules. Examples of permease and transporter ubiquitination are

found in yeast, with the general amino acid permease, Gap1p, targeted by the ubiquitin ligase

Npilp for ubiquitination and subsequent degradation in response to the addition of ammonium

(Springael and Andre, 1998; Rotin et a\.,2000). Although direct or indirect effects of CreB/CreC

on CreA are discussed above as alternatives, both models could apply as they do not conflict with

each other. ln order to further characterize the roles of CreB and CreC in the cell, we analysed

phenotypic suppressors of some or all of the phenotypes of creB and creC mulant alleles, in

order to identify proteins involved in the ubiquitination of substrates that are deubiquitinated by

the CreB/CreC complex. Genetic analysis here has shown that the acrB2 mutation is a

suppressor of the phenotypes conferred by mutations in creB and creC, including the suppression

of the derepressed expression ol alcA (encoding Adhl) in creB and creC mutant strains, as

judged by growth on medium containing allyl alcohol, These phenotypes are consistent with a

regulatory defect associated with the membrane and signalling, but not with a simple

interpretation that only permeases and transpofiers are affected, as for ethanol, a fat soluble

compound, no permease is required or exists.

fhe creD14 mutation arose as a spontaneous phenotypic suppressor of creC27 that led

to tighter repression of enzymes subject to carbon catabolite repression in the presence of

glucose, and was also shown to suppress mutations in creB and, partially,in creA (Kelly and

Hynes, 1977). These epistatic interactions ol creD with creA, creB, and creC implicated creD as

being involved in this regulatory network in an opposing process to deubiquitination, such as

ubiquitination. creD was cloned and characterized here, and encodes a protein that contains an

arrestin-N and arrestin_C domain, a PP)ff motif and two PzYY motifs, and is highly similar to the

90


Rodlp and Rog3p proteins from S. cerevisiae. The presence of two such similar sequences to

creDin the S. cerevisiae genome led to the search for another arrestin domain containing protein

in A. nidulans, which identified a gene we have designated apyA, encoding an qrrestin-N and

arrestin_C domain and !P,\f, motif containing protein. ln S. cerevisiae Rodlp and Rog3p have

been shown to interact with the ubiquitin ligase Rsp5p, and so to determine whether this

interaction occurred in A. nidulans the homologue of Rsp5p was identified, and the gene

encoding this HECT gbiquitin ligase was designated hulA. CreD and ApyA were shown to interact

with HulA via the bacterial-2-hybrid system, with a strong interaction between ApyA and HulA,

and a weaker but clear interaction between CreD and HulA. lf CreB/CreC are involved in the

deubiquitination of CreA, among other substrates, then CreD/ApyAiHulA are clearly involved in

the ubiquitination aspect of this network. The epistatic effects of creD with creB and creC are

consistent with this model, whereby the lack of ubiquitination parlially compensates for an inability

to deubiquitinate given targets. The mutant phenotype ol creD, of even tighter repression of

enzymes subject to carbon catabolite repression, could be explained according to the proposed

model of Lockington and Kelly (2002), whereby the decreased ubiquitination of CreA in a creD

mutant background results in more efficient deubiquitination of CreA by CreB in the presence of

glucose compared to wild{ype, resulting in an increase of stabilized CreA to effect repression, To

further determine the role of the newly identified ApyA and HulA proteins in this system, these

genes should be disrupted and their phenotypes analysed to see if carbon catabolite repression

is perturbed. As ApyA interacts strongly with the ubiquitin ligase HulA, it would appear that ApyA

is involved in ubiquitination and like creD, would be capable of suppressing the phenotypic effects

of the creB and creC mutations. CreD may interact more strongly with another of the six HECT

ubiquitin ligases found in A. nidulans than with HulA, and interactions with these other ubiquitin

ligases should be tested, starting with HulE, which is the only HECT ubiquitin ligase not to have a

clear orthologue in S. cerevisiae.

Arst (1981) observed that the phenotype of acriflavine resistance and molybdate

sensitivity of a strain containing lhe creD34 mutation was similar to that of strains containing

mutations in acrB. Mutations in acrB had not previously been tested for their effects on carbon

catabolite repression, and we found that like creD?4, acrB2 can suppress the derepressed

phenotype of mutations in creC, creB and partially in creA, implicating AcrB in the ubiquitination

aspect of carbon catabolite repression. AcrB was cloned and contains three membrane spanning

domains and a coiled-coil region, and thus may function in signal transduction (Boase ef a/.,

2003). AcrB was not highly similar to any known protein, and we failed to detect any protein-

protein interactions using the BacterioMatch I bacterial-2-hybrid system to screen an A. nidulans

91

Chaoter 6: Conclusions

cDNA hbrary, nor in direct testing of AcrB with CreA, CreB, CreC, CreD, ApyA and HulA. Whilst

these results indicate the lack of a strong interaction of AcrB with these proteins, these

experiments need to be repeated with the more sensitive BacterioMatch ll reporter cells to

identify any proteins that interact with AcrB, possibly through the coiled-coil region. AcrB mutants

grew poorly on a range of sole carbon sources and on some co-amino acids, indicating an inability

to derepress the enzymes required for the utilization of these sole carbon sources in the absence

of glucose, possibly due to a lack of ubiquitination. To identity the role of AcrB, overexpression of

AcrB could be investigated by transforming a construct containing acrB under the control of the

high-level constitutive promoter gpdAinlo A. nidulans. Similarly, the phenotypic effects on carbon

catabolite repression caused by increased concentrations of CreD in the cell could also be

determined in this manner. However, overexpression of mRNA may not translate to altered

protein levels, and as such Westerns would be required to establish protein levels. Presumably

increasing the amount of ubiquitination under carbon repressing conditions would result in a

decreased amount of stabilized CreA to effect repression.

De/ubiquitination networks are emerging as an essential control mechanism in the

regulation of development. A well-studied case of regulation via the de/ubiquitination of

substrates is that of eye development in Drosophila, E3 ubiquitin ligases have a number of roles

during Drosophila eye development, including control of cell proliferation, specification,

differentiation and death, For the correct development of photoreceptors to occur in the

developing eye of D. melanogaster, the Fat Facets deubiquitination enzyme is required to

deubiquitinate epsin (encoded by the liquid facets gene), thereby stabilising epsin and promoting

endocytosis, which prevents the misspecification of photoreceptor cells in the developing eye

(Chen and Fischer, 2000; Cadavid et al., 2000, Chen ef a\., 2002). ln the absence of Fat facets,

epsin remains ubiquitinated and is degraded, promoting cells to inappropriately adopt a

photoreceptor fate. A mouse homologue of Fat facets, Fam, has been identified and it has been

suggested to play a role in mouse development via its deubiquitination of epsin (Chen ef a/.,

2002; Oldhamet a1.,2002). This is one of the few cases where the deubiquitination enzyme, the

ubiquitinated substrate, and the role they play are known, although the E3 ligase that targets

epsin for ubiquitination is yet to be determined,

There have been very few cases of mutations with defined phenotypes affecting both the

ubiquitination and deubiquitination pathways of the same regulatory ubiquitination system. The

evidence reported here implicates CreD, AcrB, ApyA and HulA in the ubiquitination aspect of the

regulatory network including the CreBiCreC deubiquitinating complex that is involved in carbon

catabolite repression. CreD, ApyA and HulA, have homologues in yeast, namely Rodlp/Rog3p

92


and Rsp5p, whilst the deubiquitination aspect of this network, the CreB and CreC complex, do not

have any highly similar sequences in yeast, but do in other filamentous fungi and higher

eukaryotes such as humans, again highlighting the differences between carbon catabolite

repression in A. nidulans compared to S. cerevisiae. The linking of deubiquitination and the

regulation of carbon metabolic pathways in A. nidulans has allowed us to study the key regulatory

role of de/ubiquitination in this process, Ubiquitin and the proteasome are being increasingly

implicated in gene control, including the control of transcriptional activators by regulating their

cellular location, their association with other proteins, and their stability and thus abundance. The

clear mutant phenotypes involving creD, acrB, creB and creC represents a chance to identify

roles and substrates of a regulatory ubiquitination/deubiquitination network that may well extend

beyond carbon catabolite repression in A. nidulans.

93


94

Appendix A

APPENDIX A

The pB3 plasmid partially complemented the creD14 mutant phenotype, and in an

attempt to identify creD and the nature ollhe creD34 mutation, the pB3 region was sequenced

from both a wild{ype strain and a creD34 mutant strain (see Section 3.2.3). Custom

oligonucleotides were designed to PCR amplify this region in sections, and to sequence these

sections (see Table 4,1). The amplified sequences of individual regions were aligned to give the

complete sequence of pB3 (Figure 4,1). The pB3 region did not contain lhe creD34 mutation.

Table A.1 Custom oligonucleotides designed for the pB3 plasmid

PRIMER NAME PRIMER SEQUENCE (5'TO 3')

D1 CCCAGTCCACCAATCCTCC

D2 ATTGGCGTCCCTACGTTCG

D3 CGCTGAAGCCGCTGGTGC

D4 CGTAATGTCAACGAGGCC

D5 TGGAACTTCAGGTAACGC

D6 GAATGTGCGGACGCGGCG

D7 TGTTATGATCTGGCGACG

D8 CAGATGGCTACAGGTTGG

D9 CCTTTGCATCGAATAGGG

D10 GCCTCCGTTTGCATCAGC

D11 CTGATCGAGTAGCTTCGC

D12 GGTTGAGCTTCCAGGCGG

D13 CATTACAGCATGGACGCC

D14 GGAACTTGGTGTGGCTGG

D15a cccAcccrrccrccccrcD15b GGCATCGTTGAGTCCACC

D16 TCATGGTCCGCGCCGTCC

D17 TCCATGGTATCCTGTCCG

D18 TGATGTGGTCTGGGAGGG

D19 ATCAGGACGGTGACTTGG

D20 GAGGTGCTGATAGGACGG

D21 TAGCGTAAGGGTTCCGGC

D22 AGAACCCGGCGAGCGTGC

D23 AATCTGGAGGCGACGAGG

D24 CAGAGCATATCGTTACGC

D25 TGAGTCGTTCGGTTCGGC

D26 GGGAGGTAATAAGGCTGG

95

Appendix A

1

101392082173464r5484553

AGCTÄACCAGAÀATGGATTCACAGACGACACGTGCAATGCCGGAGACATTCAGATTGTAGATGAAGTAGTGAGATGGCGTTAAÀGACAÄÂAGGCGTTCA-AGTTCACAGGAAGGGGAGAGGAGTCTATCGACTTCCAAGCCAACGGGCAGCGGCAGTGTGGÀÀTTCCGGGGÂÀCTTGGTGTGGCTGGTTGGTGGÀÀCTTCTGCGTGGGGCAAGCTACCGATGGCGCCGACATAGTACATACATGCÀCTGACATGCCAGGAAACTATACATAGAGGCTCGTGCTTCACATTTGCCATTTTAGACTTÀ.A,TÀTTCCATTCTGGAGTGACAAÀGTGGACGCCTGTGTCAGGTTCTTAÀCCTTAÀTGGCTGTCTATTCGACAÀÀGTGCCTGTAGCTCAÀCACATTGCGAÀGATTTCATATACATTGCTCGGTATTCGCAÀT.AÀATCTATCCAÀAGCAACGCAATAÀTTGATTTCAGGGGTATCGAGTTCCATCATTACAGCATGGACGCCCTCAÃ.CCAGGCGTCAGACTCCACTACCACTGACTCGTCGCCAGATCAT

*VGSGSVRRWIMAÀCAGCAGCAGCTCCCGCACAAÀCCACAGCTCCGAGAGCAATTCCCCACGACATCCATTTGCCCTCGAC

VÀAAGÀ C VVÀ G LA I GW S MW K G E VGTCTTCCTCGCTCCGAGGGAÀTGCTGTACTCGGTTCCATTGCGGCTTCTGCGATGTGCACAAÀCACCTC

DE E S R P F ÀT S P EMA,A E A I H VF VETTGAÀTGGÀACTCCAGGTÄÀCGCTCACATGAAGCGGCGGACCAGGCATATTCAATAGAGCTGTGTATTC

QI S SWTVSVHL PPGPMNLLATYEATGAGGCTGATGTTCAGCGCGCTGGGTTGTACGGTCTAGGTCAGCCTTGAGTGCGATGTACACACTCGG

HPQ H EÀRQTTRDLDAKLAI YVS PAAGCTCCTCCAGGTGTGGATACTTCGCCCTGAGAGCGGGTATATATGCGAATGAATCGGGGTCTGACGA

LE E LH P YKARLAP I YAF S D P D S S

GTCATATGTGTATACGATCACATCACACTGATCGAGTAGCTTCGCTTGATTTTCAAGAÁ.TCGCAGGTTCDYTYVI VDCQDLLKAQNE L I A P E

TAÀ.CTCGCCGAGCTCATCCATGATCAGGTAACATTGTTTTCCGCCTGGAAGCTCAÀCCGTGTTGACTGCLEG LE DM I LYCQKGG PL EVTNVA

GGTACGCGGTTGAATTGTGGGGTGATAAGTGGTACTAA.A.TCCGCGGGACAGÄAÀ.A,GCATCGAGÀÀGGGCTR P Q I T PH YT T S F G R S I, F AD L LA

AGATTTGCCAGACCCCGGAGCTCCGACTATGTGGCCAAGGACGACATTGCGACCGACGCGTCCTGGACGS K G S G P.A G V I H GLVVN R G VR G P R

TTTTCTCCGCTTACGGGGTCGTGTTACCTTAAGAGCCGCGGTCGTGGAGGGGTTGCTCCGATCTGAAGAKRRKR P R TVK LAÀTT S PN S RD S S

CTCGAATCCCAAGTACGCTAGATATTCCAGAGTCGTCTTGGGAGAÀGTAÀAGGTTGTCATACTCCACTGEFG LYALYE LTTKPSTFTTMSWO

TGCTAGCCAGCCCTGAÀGAGTGACATGGCCAGCCTCGTTTCTGACAGTGCATGAAGGAÄÀTGAGCCGTCALWG Q L TVHGAENRVT C S P F S G D

AGCCCAAGAGGCGGGCAATCCAGGGGTGGGTGCGAACAATGATGCTAÀCTCGGCATCGTTGAGTCCACCAW S A PL G PT PAFL SAL EADNLGG

ATCATTGTCTTTGTCTGAGAGAA.GGAAÀAGGTTGACAAAGAÄCCGATAGCCCTCAGGAGACAÀTTCCGCDNDKD S LLF LNVF FRYG E P S L EA

TGATGCAÀACGGAGGCACCTCAÀACTTTGGGTGGAGATAGCTCTCTTGCÀACGAÀAGGCTATCGGTGTASAFPPVEFKPHLYSEOLSLSDTY

CTGAÄATGCGCGAÀGTATGATCCAÄÄCTGTTTCATGGCGTCCTTTCTCGGCATACATCTTGTTCAGATG

QFARL I I WVTEHRGKEAYMKNLHAÀTAÀÀTCCCCGGCAÄTCTATCCCGGACGGCGTCACAGAGTCAGGATGTGTTTTCTGAÀTTGTCTCTTT

IFGRCD T GS PTVSDPHTKQ I TEKTATATGGACCAÀGTCTTCTTCGCTCAGAGGTTTCTCAÀÀÀCACCTCATCTGÄÀAATCCTTTATCTCTTT

I HVLDE E S L PKEFCRMQF D K I EKATCTGAGAGATAÀCCGTCCCGGTCTTTATCACTGAGGTAGÀAGATGCGTTGTAACGCGGCGACAGCCGC

D S L YGD RDKDS LYF I RQ LAAVAACGGTTTTÄÀGGCTGATTCCTTTGCATCGAATAGGGGTGCGATAGGGTGTGTGACCGCTTTCTGGCAAAG

PKLA S E KADF L PAI PH TVA KQ C LGAÀÄAÀGGCCTCGTTGACATTACGATGTTCTCGGGCACTGGTCCGAATGCATGAATCGATTTCCTTGAÄ

F FA ENVNRH E RASTR I C S D I E KECTCTGACATCÀÄCGGCAACATCTCGTCCTCGATGACTTGCGTCTCGGTATGATCGGCAGCAAGATCGGA

E SM L P I,M E D E I VQ T E T H DÀÀ L D S

CTTGTTGGCACACAGCACGACAGGCACATTCACGCCGAGAGAGCGGÀÀATAGGGCAGCCAGAAÀAGCGCK NA C L VV P VN V G L S R F Y P L VÙ F I, A

AACCCTCTCATAGCTATAGTGATCAGAATAGACAÀGCA-ATATCACGTTCGATTTCCGAATTTCTCTAGCVRE Y S Y HD S YVLL I VN S KR I E RA

CAÀGTTGCTCCGTTCTTGCGGCACAGCAGAGGTATCGACCACCGTTGTCGTTGTAACATTCTCGGGGGTLNS RE Q PVA S T DVVTTT TVNE PT

TCCAATGGTAGGAGGGATAGTGATCTGTGGCAGAATGGGCTGGATCTTGTTCGTCACAAACACGCCTTTGT T P P I T I Q PLI PQ I KNTVFVGK

TACGAGGGAGGTÀATÀAGGCTGGACTTTCCAGTTCCCTCATCCCCGCAÀA.CACÀAATTCGCAc I agaTgVLSTILSSKGTGEDGCVCIR

qtgtcagtatc t t tgt tc tttcgtggacqcc tcaacaaaaqagagc tccaaCCTGTAGCCATCTGAACAVTAM

AATAÀAGAÀGCACTTGAÄ.GAAGCGAÀTTCTAAGGCCCTCTGAGATCATTGCTGAACTTGGAGTACACCCTACACCGCCATGAGGCCAGGAGGCTGGCGCGTCAGTTCAGGTTAGGAACCAGAGCAGCTAGCAÀGTCCGACGGÀAGTTTGGGCGATCGCCGCGGTCTTGTGGCGGTGCGATGCGTTTAAGCCTCAGGCCTCCAAATGGACAAGCTTGACACACATTCGGGACCAÀCATGAGCCTTGGTCTCAGTGGGTGACCAGCATACTGCAACAGAGCATATCGTTACGCTATTTCAATATGGGACTAGATTAATCTGTATTCTGTACCACATGCCGCGCCCGCCCÄÂÂGATGATGTAÀCGGAGTTAGCCAAGTGCCGAÀCCGÀACGÀCTCÀGCCÀCÀÀTCTGGGCCATCTAC

622

69r

160

829

898

96'/

103 6

110 5

lLl 4

L243

L3L2

l3 B1

14 50

1519

15BB

165't

1726

L't 95

1864

1933

2002

207 r

2L40

2209

227 B

23 41

24L6

2485

623

600

517

554

531

508

485

462

439

416

393

370

341

324

301

218

255

232

209

186

163

140

tL1

94

]L

48

25

6

I25542623269227 6L28302899

96

2968303731063L1 5

3244

CCGATTTTACGAGAAGCTTATGGACACCACTTCGTAGTTGCTATCCTTAAGCATAGCTGCTTGAÂGACTGTÀTCGCCATCTGTCTATAGTTCCCCCTGTGAAGCGCAGTACTGAÀCGGGGAAATCAGCGGCTCAGCCTAGCATCCATTCTTAGCTCCACTTCTAGGCCCTCCCATTTGGCÀÀGGCCTA.ACGTCGAÀCATÀÀTATTACAGTTTCGACAACGTTCGAAACTCTCCCCAGCCTTCCTCGCCTCTTTACÄÀCAACCATTCCCTCÄÀGAÀCCCGGCGAGCGTGCGCAÀGATGCCTCTTGGAÀTTCACAÀCCCTCTCCCTTCGTCGCTATCGAgTaagIcc

MPLGIHNPLPSSLSStcgtcqcctccagatttccccgcagt.tacccacaggcgacaac LtgggcgLgagc tgac tgctaatcctcaatttcEc tcggtagGCGAATGCÀAAAAGGCTGGCAÃÀÀTCCTAGCATCTTTTGTTGACCCGCGGCAA

ECKKAGKILASFVDPRQGCATTCGGGCCTGACAAGGTCATCCCCCCTGAGGTATTAGCTGGTGCTÄÀGg Eacgcaaaggalac IcgIAFGPDKVIPPEVLAGAKqtgaagc LgaLaqc tgaattatgqctgattgcttgrgcttctagcGTCTTGCCATTTTGACCGTCCTGAA

GLAILTVLKAGCAGGGTTTTTGGGTTCCGGTCGATTTGGTTCGGGTATCGTCGTTGCTCGACTAGCTGATGGATCATGAGF LG S GRFG S G I VVARLADG S W

GTCCGCGCCGTCCGCGATCGCÀACCGCAGGTGCTGGATTTGGAGGTCAGATTGGATTTGAGTTGACCGASA P S A I ATAGAG F G G Q I GF E IJ T D

TTTCGTCTTCATCTTGAÀCGATGCTGCCGCCGTCCGCACATTCTCTCÄAGCCGGAÄCCCTTACGCTAGG

AGGTAATGTTTCGATTGCCGCTGGGCCGGTTGGTCGAAACGCTGAAGCCGCTGGTGCTGCTAGTACGAAGNVS I AAGPVGRNAEAAGAA STK

GGGTGTGGCCGCTGTATTCTCATATTCCAAÀACCAAGGGACTTTTCGCCGGTGTCAGCTTAGAÀGGAÀGGVAAVF S Y S KT KGL F AGVS L E G S

CATGCTGGTCGAACGTAGGGACGCCAÀTGAGAGACTCTACAATAGCCGAGTATCTGCTCGCCAGCTCCTM L VE RR DAN E R L YN S RV S AR Q L I,

CAGTGGTACTATCCCGCCTCCACCCGCCGCCGÀACCTCTAATGCGTGTGCTGAÀCTCCCGCGCTTTCTAS G T I P P P PAAE P LMRVI,N S RAF Y

CGGCGTGCGCACAÀACGGCGACTCTATGTACAÀCGATATCCCCGTCTACGATGATCGTCATGATGATGTGVRTNGDSMYNDIPVYDDRHDDV

GGTCTGGGAGGGTCGCAGGGGAGAGGCATATGGCGAGGGGATCAGACGTGATCGGACAGGATACCATGGVW E G RRG E AY G E G I RRDRTGYH G

ACCTTCAGATGACTACGAATACCACGACCGGCCGCGCCGTGCAACGACGTGGGCTGACGATATATATGAP S DDYEYHDR P RRATTWADD I YD

TCGTCCTGCTGGAGGCTTGAGTCGTTCCTCCACCGCCCGATTTAGCAGCCGCAACGACACCTTTGATACRPAGGLSRS S TARF S SRNDTFDT

CTACAATAGACAGCGGAGCAÄCACCTATGATGATGATTATGTGTACTCTGACCGCAAACCCAGTCGTCCYNRQRSNTYDDDYVYSDRKP SRP

TACAGCACCCAAGCCCgTT tcgacagcgcactggcaggccgtccgt tacgcqagGACCAGGCCATTGCGTAPKPDQAIA

CTGTACACATTCGACGCGGATCAGGACGGTGACTTGGGTTTCAAGAAGGGGGAGATTATCACAÀTCATCIJ Y T F DAD Q D G D L G F KKG E I T T I T

AAACGCACCGAAÀÀGAAGGAGGATTGGTGGACTGGGCGGATCGGTGACCGCGTCGGCATATTCCCTGCGKRTE KKEDWWT G R I GDRVG I F PATAAGATACCTTGCTCTGA.AÀTGGCTGGACTGCTTGCTÀÀCTCAGTGCCTTTAGGAÀTTATGTTGACGCA

GCTTÀAGACTTTCGCCCATGACCATTTATTTATATGCAACGAAATTCATGACCTTGCCCGCTTATACCTCTCCTTTGGCAATTTTCATTTATACCGATCGAGTGTGTTTCAGTCTTTTTTTGGAATCCTTGGGCATTTTATCCCATCTCGTCCGAGCTTCTCTTTTCAGCTCTTGCGTAAGCATTGGAGTTTGAAGGTGGTTTTACGAGTGCGTACAGGCGA.AAGAGAATTCGGCGCTTCTCAGAATATCÀAGTTCAAATTCCCCTGCCTTCCCACTTATTATCCGTCCTATCAGCACCTCTAGCAGCTCTTGTGATTATTATTACTGTCCAATATACAGGTTTTCTTTGATACAGGCTTGTCTCGGAGAÀÀTT.AÀCATTÀÀÀGAGGTAGATÀAGCGTCATCTGCAGGTTATTCTCGCAGTCGTTGAGGCTGGGACCCGTCACCTGCAAGGAGAGGAGATCT

1533133382

3 45'l-

3520

3589

3658

3121

319 6

3 865

3934

4003

4012

4]4t

421-0

421 9

4348

44r7

4486

4555

4624

F V F I L N D AAAVR T F S Q AG T I, T L G

32

49

58

8L

104

L21

r_50

Il3

196

2L9

242

265

288

311

334

344

-7t> I

390

39146934't6248314900496950385l_07

Figure A.1 Nucleotide sequence of the pB3 plasmid, The amino acid sequences of the

Gonlp-like protein, highlighted in grey, and the Ysc84p-like protein are shown in single

letter code beneath the DNA sequence, with numbered amino acid residues indicated on

the right-hand side, and numbered nucleotide positions indicated on the left-hand side.

Eco?lsites are underlined and bolded. Putative introns are in lower case letters,

Appendix A

97

Appendix A

98

References

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Addendum

Corrections:

Page 38 section 3.2.3 Line 23 "suppressed" shourd replace "complemented".

Additions:

Page 7, Section 1.3.1 ln S, cerevisiae, Bgrlp is a component of the yeast mediator complexrequired for the transcriptional regulation by RNA polymerase ll. [Li, y:, Bjorklund, S., Jiang,Y.w., Kim, Y.J., Lane, w.s., stillman, D.J., and Kornbery, n.o. (lggs) yôast gbbãitranscriptional regulators Sin4 and Rgrl are components of mediator complex/RNÂ po$merãse llholoenzyme ' Proceedings of the Nationat Academy of Sciences of the Ùnfted Sfafes of America92:1 0864-1 08681.

Page12, Section 1.3.5 PEST sequences are hydrophilic regions rich in proline (p) glutamic acid(E) serine (S) and threonine,(T) flanked by positively charged residues that are inôrlgnt to targetsequences for degradation via the 265 proteasome.

Page 13 Figure 1.3 PrnB is the proline permease encoded by the prnB gene in A. nidutans. Thegenetic evidence suggests that the CreB/CreC complex also affects permóases (eg. prnB).

Page 53 Section 3.6.2 The C2 domain is a Caz*-dependent membrane-targeting module found inmany cellular proteins involved in signal transduction or membrane traffickìng, ánd is involved inmembrane targeting processes such as subcellular location and calcium-depéndent phospholipidbinding [Daveletov, B. A and Sudhof, T. C (1993). A single C2 domain from synapiotagmin iissufficient for high affinity Ca2+/phospholipid binding. Journat of Biotogicat Cnemistry2g6;263g6-263901.

Page 64 Section 4.3 The weak suppression of lhe creA204 mutation by the cre}s4mutation wasnot able to be detected on plate tests, but was determined by specific ênzyme assays (Kelly andHynes, 1977).

Chapter 6 The model for carbon catabolite repression proposed by Lockington and Kelly (2002,see Figure 1.3) can now be expanded to include CreD, ApyA, HulA anã AcrB. ft häs beenproposed that the CreB/CreC deubiquitination complex acts on ubiquitinated CreA to stabilize oralter the function of CreA as well as on other targets. CreD and ApyA have been shown tointeract with the HECT ubiquitin ligase HulA, and these proteins likeiy form the "ubiquitinatingcomplex" that is responsíble for the ubiquitination of CreA. The AcrB protein is also implicated iñthe ubiquitination aspect of this regulatory network, and the three transmembrane domains implya role in signalling. Thus mutants in both the deubiquitination and ubiquitination aspects of thisnetwork have been isolated as a result of their mutant phenotypes. To test this model, the abilityof the CreD/ApyA/HulA complex to ubiquitinate CreA and othei substrates should be tested. Thiiwould require the isolation of a specific antibody to CreA (which so far has been unsuccessful)toidentify CreA. This would be used to determine the presence or absence of ubiquitinated CreÁ increD, apyAand hulA mutant backgrounds, thus testing the model.

the role of the acrb and cred genes in carbon catabolite … · 2015. 4. 22. · agda, an...

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